From the School of Cell and Molecular Biosciences,
The Medical School, University of Newcastle upon Tyne, Newcastle upon
Tyne, NE2 4HH, United Kingdom, the § Oral Infection and
Immunology Branch, NIDCR, National Institutes of Health, Bethesda,
Maryland 20892, and the
Defense Science and Technology
Laboratory, Porton Down, Salisbury SP4 OJQ, United Kingdom
Received for publication, February 5, 2003
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ABSTRACT |
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Protective antigen (PA) is a component of the
Bacillus anthracis lethal and edema toxins and
the basis of the current anthrax vaccine. In its heptameric
form, PA targets host cells and internalizes the enzymatically active
components of the toxins, namely lethal and edema factors. PA and other
toxin components are secreted from B. anthracis using the
Sec-dependent secretion pathway. This requires them to be
translocated across the cytoplasmic membrane in an unfolded state and
then to be folded into their native configurations on the
trans side of the membrane, prior to their release from the
environment of the cell wall. In this study we show that recombinant PA
(rPA) requires the extracellular chaperone PrsA for efficient folding
when produced in the heterologous host, B. subtilis;
increasing the concentration of PrsA leads to an increase in rPA
production. To determine the likelihood of PrsA being required for PA
production in its native host, we have analyzed the B. anthracis genome sequence for the presence of genes encoding
homologues of B. subtilis PrsA. We identified three
putative B. anthracis PrsA proteins (PrsAA, PrsAB, and
PrsAC) that are able to complement the activity of B. subtilis PrsA with respect to cell viability and rPA secretion, as well as that of AmyQ, a protein previously shown to be
PrsA-dependent.
Bacillus anthracis is the etiological agent of anthrax,
an historically well documented disease. Anthrax affects mainly
herbivorous animals such as sheep, cattle, and wild herbivores,
although all mammals are susceptible. The bacterium is transmitted
predominately via spores rather than vegetative cells.
Infection is usually acquired through the uptake of spores from soil or
infected animal products by inhalation, ingestion, or cutaneous
abrasions. Fully virulent strains of B. anthracis possess
two major virulence factors, the anthrax toxins and a The anthrax toxin proteins, edema factor, lethal factor, and protective
antigen (PA)1 are encoded,
respectively, by genes cya, lef, and
pag, located non-contiguously on plasmid pXO1 (185 kbp)
(1). PA binds to either edema factor or lethal factor to
produce the binary edema or lethal toxins, respectively (5, 6). Mice
challenge experiments with isogenic strains of B. anthracis expressing either lethal toxin or edema toxin
indicate that, although these toxins act synergistically, lethal toxin
is the key virulence factor (1).
The other main virulence factor, the The current UK and USA human anthrax vaccines, although differing
slightly, are based on PA (83 kDa.). The UK anthrax vaccine consists of
an alum-precipitated culture filtrate from an aerobic static culture of
B. anthracis strain Sterne 34F2, whereas the USA vaccine
consists of an alhydrogel-adsorbed cell-free culture filtrate of
B. anthracis V770-NP1-R, grown anaerobically in a fermenter.
Both strains, although avirulent, must nevertheless be handled as class
III pathogens. In an attempt to reduce the costs associated with
handling strains of B. anthracis (5, 6), various alternative
production systems have been explored. However, with the notable
exceptions of Bacillus subtilis and Escherichia coli, these have met with little success (7, 8). Cloning pagA into B. subtilis strain IS53 (9)
resulted in the secretion of recombinant PA (rPA) to a concentration of
about 40 µg/ml, ~3-fold higher than that obtained with B. anthracis Sterne (~15 µg/ml). Strain B. subtilis WB600, a multiply extracellular protease
deficient-strain (10, 11), has been used to reduce degradation of rPA
by co-produced proteases.
In an attempt to increase rPA production from B. subtilis, we have examined the role of PrsA on rPA secretion. PrsA
is an essential lipoprotein component of the B. subtilis
protein secretion pathway, where it functions on the trans
side of the cytoplasmic membrane as a post-translocational folding
factor (12). PrsA has been shown to be rate-limiting for the high level
secretion of Although the B. subtilis YacD, a PrsA paralogue, is not able
to complement PrsA activity, we show here that three B. anthracis PrsA orthologues are able to do so with respect to
both viability and protein secretion in B. subtilis.
Bacterial Strains, Plasmids, and Growth Conditions--
Table
I lists the bacterial strains and
plasmids used. Strains were grown and maintained in Luria-Bertani (LB)
medium (per liter: 10 g of tryptone, 5 g of yeast extract,
10 g of NaCl), excepting for the determination of rPA production
from strains MFJ683, MFJ943, and MFJ945, which were grown in complete
anthracis medium (per liter: 35 g of tryptone, 5 g of yeast
extract, 6 g of Na2PO4.7H2O,
1 g of KH2PO4, 5.5 g of NaCl, 40 mg
of L-tryptophan, 40 mg of L-methionine, 5 mg of
thiamine, 25 mg of uracil). Antibiotics were used at the following
concentrations: ampicillin, 50 µg/ml; chloramphenicol, 5 µg/ml;
erythromycin, 1 µg/ml; kanamycin, 10 µg/ml. Unless stated
otherwise, isopropyl- DNA Manipulation and Strain Construction--
B.
subtilis was transformed with plasmid DNA using the
"Groningen" method (14). pRCW101 was constructed by amplifying a fragment (~300 bp) at the 5'-end of prsA, including
ribosome binding site, from chromosomal DNA using oligonucleotide
primers (Bsu-prsA-fwd 5'-CGCAAGCTTATTTGGAATGATTAGGAG-3' and Bsu-prsA-rev
5'-CGCGGATCCAGGGCAGTATATTGATCG-3') with restriction endonuclease
sites (BamHI and HindIII) incorporated at their
5' termini. The fragment was cloned into pMUTIN4 (15) and, following
recovery in E. coli, pRCW101 was integrated into the
B. subtilis chromosome via a single crossover
recombination at prsA. The resulting strain, RCW201, had a
truncated non-functional version of prsA under the control
of its native promoter and an intact copy of prsA under the
control of the Pspac promoter.
Plasmids encoding B. subtilis (Bsu) and B. anthracis (Ban) prsA genes, pRCW207
(Bsu-prsA), pRCW208 (Ban-prsAA), pRCW209
(Ban-prsAB), and pRCW210 (Ban-prsAC) were
constructed by amplifying the genes and associated ribosome binding
sites from chromosomal DNA using oligonucleotide primers with
restriction endonuclease sites (BamHI and
HindIII) incorporated at their 5' termini
(Bsu-prsA fwd,
5'-CGCAAGCTTATTTGGAATGATTAGGAG-3'; Bsu-prsA rev2, 5'-CGCGGATCCAAGCCGCAGTTCTCAGCA-3';
Ban-prsAA fwd3, 5'-GCCGAAGCTTGTAGGAGTGTTTATCGAA-3'; Ban-prsAA
rev4, 5'-CGCGGATCCATACAAAAAAAGCTCGG-3'; Ban-prsAB
fwd3, 5'-GCCGAAGCTTTAACATATTCGGATGAGG-3';
Ban-prsAB rev4, 5'-CGCGGATCCAAGCACAACCTTATCTG-3';
Ban-prsAC fwd5,
5'-GCCGAAGCTTGTGAGGTATTTTGAATTG-3'; Ban-prsAC
rev6, 5'-CGCGGATCCCCCTTTTCTAATTACAG-3'). The amplified fragments were
cloned into pJPR1 using the multiple cloning site located immediately
downstream of the Pxyl promoter. Adjacent to the cloned
prsA gene is a chloramphenicol resistance gene, and this
region of the plasmid is flanked by the 5'- and 3'-ends of B. subtilis amyE. Strains in which plasmids pRCW207, pRCW208, RCW209,
and pRCW210 had integrated into the chromosome of RCW201 via
a double crossover recombination at amyE were isolated after selection of plates containing chloramphenicol.
Western Blotting and Dot-blot Analyses for the Quantitation of
rPA--
For Western blotting, proteins separated by
SDS-polyacrylamide gel electrophoresis were transferred to a
nitrocellulose membrane (Protran, Schleicher & Schuell UK Ltd, London)
using a mini-trans-blot system (BioRad). For dot-blotting,
samples of culture supernatant were transferred to a nitrocellulose
membrane using a vacuum manifold (Invitrogen). Proteins were detected
with in-house-specific rabbit anti- PA antibodies and swine
anti-rabbit IgG horseradish peroxidase conjugate (DakoCytomation Ltd,
Ely, Cambridgeshire, UK) and visualized with the colorimetric
developing agent 4-chloronapthol. The images generated by Western or
dot-blotting were analyzed with QuantityOne (Bio-Rad). Background
values were removed prior to determining the intensities of individual
protein bands. The relative band intensities were compared within and
between lanes on a single gel.
Determination of Multiple Alignment of Protein Sequences--
The protein
sequences were aligned using the ClustalW tool
(www.ebi.ac.uk/clustalw). Calculation of the relative identity of the
sequences was carried out using the Needleman-Wunsch global alignment
algorithm, which is available within the EMBOSS suite of software at
the Human Genome Project Resource Centre (www.hgmp.mrc.ac.uk).
Influence of PrsA on rPA Secretion--
Mutants of prsA
encoding a defective PrsA protein exhibit a defect in the secretion of
AmyQ, an
To confirm that PrsA influences the yield of rPA, strains were
constructed so that the level of PrsA could be controlled. The pMUTIN4
integration vector (15) was used to construct B. subtilis strain RCW201 in which expression of prsA
was under the control of the IPTG-inducible Pspac promoter.
Because prsA is essential for viability (12), we confirmed
that RCW201 had an absolute requirement for IPTG. At IPTG
concentrations between 0.1 and 10 mM, the growth rate and
yield were similar to that of the wild-type strain. However, at lower
concentrations, growth was characterized by a lag of several hours and
the growth exclusively of IPTG-independent suppressor mutants. In
contrast, the growth of a strain in which the prsA homologue
yacD was placed under Pspac control was not
IPTG-dependent (data not shown).
RCW201PA (i.e. RCW201 with pPA101) was used to analyze the
relationship between prsA expression and the yield of
secreted rPA. RCW201PA was grown in LB broth in the presence of various concentrations of IPTG (0.5-10 mM). rPA production was
determined by quantitative Western blotting analysis of culture
supernatants. The data (Fig. 2) show a
direct relationship between the level of prsA expression
(i.e. IPTG concentration) and the yield of mature
rPA.
B. anthracis Possesses Three PrsA Homologues--
The unannotated
B. anthracis genome sequence (The Institute for Genomic
Research: www.tigr.org) was searched for homologues of B. subtilis prsA. The genome sequence was translated in all six
reading frames and protein-protein alignments were carried out using
the blastp algorithm (18). Three B. anthracis proteins, named PrsAA, PrsAB, and PrsAC, showed homology to B. subtilis PrsA. Alignment of the B. subtilis and
B. anthracis PrsA homologues indicated a high
degree of sequence conservation throughout their entire lengths (Fig.
3). The relative identities of B. anthracis PrsA homologues with B. subtilis
PrsA ranged from 38-44%, and the Ppi PPIase I and II domains were
well conserved. In contrast, the relative identity between B. subtilis PrsA and YacD was 24%; no YacD homologue was identified
in B. anthracis.
Of the three B. anthracis PrsA homologues, PrsAA shows the
highest similarity (44%) with B. subtilis PrsA. Analysis
of the region of the chromosome either side of prsAA
revealed the presence of three genes, yhaI, hpr, and
yhaH, that co-localize with prsA of B. subtilis (Fig. 4). No genes
co-localizing with B. subtilis prsA were found in
the chromosome regions either side of the B. anthracis prsAB
and prsAC genes. This suggests that B. anthracis prsAA is the primary homologue of B. subtilis
prsA and that B. anthracis prsAB and
prsAC were acquired after these species separated from a
common ancestor.
B. anthracis PrsA Homologues Are Functional in B. subtilis--
The B. subtilis PrsA homologue YacD is
not able to complement PrsA activity with respect to either viability
or secretion (data not shown). We therefore determined whether the
B. anthracis PrsA homologues could function in
B. subtilis by establishing a complementation
system in which B. subtilis and B. anthracis prsA genes could be expressed independently. The basis of this complementation system is strain RCW201, in which expression of the
native prsA is under the control of the IPTG-inducible
Pspac promoter. Introduction of xylose-inducible copies of
the B. anthracis homologues Ban-prsAA,
Ban-prsAB, or Ban-prsAC into the chromosome of
RCW201 at the amyE locus (encoding a non-essential
The ability of individual B. anthracis prsA orthologues to
complement the essential activity of B. subtilis prsA
was examined on agar plates, using the strains described above, in the
presence of IPTG and/or xylose. The results (Fig.
5) indicated that all three B. anthracis prsA homologues are able to complement B. subtilis prsA with respect to viability. Moreover, the simultaneous
expression of both genes was not detrimental to growth, indicating that
the B. subtilis and B. anthracis PrsA proteins do
not interfere negatively with each other. Colonies appearing on media
lacking both IPTG and xylose were found to be IPTG-independent and were
most probably suppressor mutants (19).
Strain RCW303, carrying Ban-prsAA, exhibited smooth colony
morphology in the presence of xylose, conditions favoring
Ban-PrsAA production. This compared with a rough morphology
when the host PrsA was produced or co-produced with PrsAA either in the
presence of IPTG alone (Fig. 6) or with
IPTG and xylose (not shown). In contrast, wild-type B. subtilis and the strains carrying Ban-prsAB, Ban-prsAC, or a second copy of Bsu-prsA exhibited
a normal rough morphology in the presence of xylose (Fig. 6) and/or
IPTG (not shown). These observations indicate that Ban-PrsAA
has a different substrate specificity to that of the other PrsA
proteins.
The Influence of B. anthracis Homologues on the Production of AmyQ
and rPA--
B. subtilisPrsA levels influence the yield of AmyQ (12)
and rPA (Fig. 2). Consequently, strain RCW302 with a xylose-inducible copy of Bsu-prsA and strains RCW303, RCW304, and RCW305 with
xylose-inducible copies of Ban-prsAA, Ban-prsAB, and
Ban-prsAC, respectively, were used to determine their
influence on the secretion of AmyQ and rPA. These strains were
transformed with either pKTH10, encoding AmyQ, or pPA101, encoding
rPA.
The B. subtilis prsA complementation strains transformed
with pKTH10, viz. RCW302Amy, RCW303Amy, RCW304Amy, and RCW305Amy, were
grown in LB broth containing 1% xylose. The growth of the strains
expressing Pxyl-controlled B. subtilis or
B. anthracis prsA genes was similar to that of RCW101
expressing B. subtilis prsA from its native promoter (Fig.
7). In each case, AmyQ synthesis was
induced during transition from exponential to stationary phase and the
highest yield of AmyQ was observed in stationary phase. The stability
of AmyQ in culture medium was confirmed by the maintenance of high
Equivalent experiments were carried out with strains producing rPA,
RCW302PA (with Bsu-prsA), RCW303PA (with
Ban-prsAA), RCW304PA (with Ban-prsAB), RCW305PA
(with Ban-prsAC). Again, the growth of strains expressing
Pxyl-controlled B. subtilis and B. anthracis prsA genes was similar to that of RCW102 in which the
B. subtilis prsA was expressed from its native promoter
(Fig. 8). In all strains, rPA production
peaked during transition to the stationary phase but then declined with
different kinetics, presumably because of the presence of proteases in
the culture medium (20). As was seen for AmyQ, the highest rPA yield
was observed when B. subtilis prsA was expressed from its
native promoter, in RCW102, whereas the maximal rPA yields of strains
expressing B. subtilis or B. anthracis prsA genes
from the Pxyl promoter were between 2- to 3-fold
lower. Strains expressing Ban-prsAA and Ban-prsAB produced slightly higher peak yields than those expressing
Bsu-prsA or Ban-prsAC.
Lipoprotein PrsA, essential for the viability of B. subtilis, is an extracellular chaperone involved in the
post-translocational folding of specific secretory proteins. To date,
the only secretory protein that has been experimentally demonstrated to
require PrsA for folding is AmyQ, and mutants with reduced amounts of
PrsA show lower We investigated the role of PrsA in the secretion of B. anthracis PA, a key component of edema and lethal toxins.
Initial studies, using the prsA3 mutation that produces
~10% of the wild-type activity, indicated a role for PrsA in rPA
production; unlike the wild-type control, no rPA was detected in the
culture supernatant of the prsA3 mutant (Fig. 1). The
importance of PrsA on rPA production was confirmed using a strain in
which the level of prsA expression induced from the
Pspac promoter was controlled by the addition of IPTG. A
5-fold increase in the concentration of IPTG in the medium (0.5-2.5
mM) resulted in a 2.5-fold increase in rPA production (Fig.
2). Even at 10 mM IPTG, the amount of rPA was only about half that observed when Bsu-prsA was expressed from its
native promoter (data not shown). Together these data indicate that the manipulation of PrsA synthesis could be a useful strategy for increasing the production of rPA.
Because rPA production in B. subtilis was
PrsA-dependent, we were interested to establish whether its
native host, B. anthracis, encoded a PrsA homologue.
Analysis of the raw DNA sequence data unexpectedly revealed the
presence of three PrsA homologues in this bacterium, PrsAA, PrsAB, and
PrsAC. Using a complementation system in which the synthesis of the
native B. subtilis PrsA or a B. anthracis
homologue could be controlled independently, we were able to show that
all three orthologues were functional in B. subtilis with
respect to cell viability and protein secretion.
These observations raise the question as to why B. anthracis
produces three PrsA proteins and B. subtilis produces only
one. An explanation might be found in the smooth colonies observed in
B. subtilis expressing Ban-prsAA rather than the
rough morphology typical of wild-type B. subtilis expressing
Bsu-prsA or one of the constructs expressing
Ban-prsAB or Ban-prsAC. The simplest explanation
is that Ban-PrsAA is inefficient at folding a specific PrsA-dependent protein required for normal cellular
morphogenesis. It also suggests that individual PrsA proteins are
specific for, or can distinguish between, different secretory protein
substrates. We attempted to test this hypothesis by analyzing the
efficiency with which each PrsA protein was able to function in the
secretion of two PrsA-dependent proteins, namely AmyQ and
rPA (Figs. 7 and 8). When individual B. anthracis and
B. subtilis prsA genes were put under the control of the
xylose-inducible Pxyl promoter, AmyQ yields were similarly
high in strains expressing Bsu-prsA and Ban-prsAB, whereas yields from strains expressing
Ban-prsAA and Ban-prsAC were significantly lower
(Fig. 7). Interestingly, the PrsA homologue that appeared to be the
most effective in mediating high AmyQ yields, PrsAB, has the least
amino acid similarity to B. subtilis PrsA.
We were not able to ascertain whether the amounts of PrsA synthesized
by each of these strains were the same, because the only antibodies
that were available were to Bsu-PrsA. Although it is
reasonable to assume that homologous genes expressed from the same
promoter produce similar amounts of their protein, the variations in
AmyQ production may reflect differential amounts of functional PrsA
because of factors such as (a) variations in mRNA
half-lives of the various prsA genes, (b)
variations in the recognition efficiency for the various
prsA ribosome binding sites, and (c) differential
stability of the various PrsA proteins in B. subtilis.
Alternatively, the various PrsA lipoproteins may exhibit differential
substrate specificities with respect to AmyQ.
In a similar set of experiments, rPA yields were examined under
conditions in which the various prsA genes were induced with xylose (Fig. 8). rPA levels were generally higher with
Ban-prsAA and Ban-prsAC and lower with
Bsu-prsA and Ban-prsAB,
although the differences in yields were not as marked as with AmyQ.
Interestingly, the kinetics of rPA degradation in strains expressing
the various prsA genes followed different patterns, most
likely because of their differential effects not only on the folding of
rPA but also on that of the host extracellular proteases that are
co-produced in stationary phase. The data (Fig. 8) suggest that the
PrsA protein encoded by Ban-prsAA increases the
persistence of secreted rPA in the medium to a greater extent that
either Bsu-prsA or the other B. anthracis proteins. The decrease in rPA from the maximal concentrations at transition phase is in contrast with the stability and persistence of AmyQ (Fig. 7).
The data for AmyQ, rPA, and cell morphology provide clear evidence that
the B. anthracis PrsA homologues show different but overlapping substrate specificities. A similar observation has been
reported for other components of bacterial protein secretion pathways.
A recent analysis of completed bacterial genome sequences (21) has
identified nine bacterial species that possess two homologues of SecA:
B. anthracis, Listeria monocytogenes,
Listeria innocua, Mycobacterium leprae,
Mycobacterium smegmatis, Mycobacterium tuberculosis, Staphylococcus aureus,
Streptococcus gordonii, and Streptococcus
pneumoniae. Interestingly, all nine are Gram-positive bacteria
that colonize or cause disease in human hosts. That study suggested
that L. monocytogenes SecA2 might be
involved in phase variation, and secA2 mutants showed a
rough, rather than the usual smooth, colony morphology and a greatly
increased LD50 compared with wild-type. At least four of
these bacterial species, B. anthracis,2 S. aureus, S. gordonii, and S. pneumoniae
(22), also encode a second homologue of SecY, namely SecY2,
indicating that they have a specialized transporter, SecY2-SecA2, for
the export of a subset of secretory proteins. In the case of S. gordonii, this secondary transporter is required for the transport
of a large serine-rich surface protein (GspB) that contributes to
platelet binding. Similarly, B. subtilis encodes five signal
peptidases, SipS, SipT, SipU, SipV, and SipW (23). Although all are
able to process secretory preproteins, only SipS and SipT are essential for viability; viability is maintained in the presence of either SipS
or SipT but not when both are deleted (24, 25). The remaining signal
peptidases have a minor role in protein secretion. The endoplasmic
reticulum (type SipW, for example) appears to be required for the
processing of two spore-associated preproteins, namely pre-TasA and
pre-YqxM (26, 27). Again these data indicate that, where they occur,
paralogous components of secretory pathways are required for the
processing of a subset of protein substrates.
In relation to the B. anthracis PrsA homologues, two
questions arise. First, when and, in the case of spore-associated
proteins, where are these proteins synthesized? Second, are individual
PrsA proteins associated with specific translocators such as SecY-SecA, SecY2-SecA2, or, possibly, even the twin-arginine transporter (28,
29), the genes for which have also been putatively identified in
B. anthracis?2
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-polyglutamic
acid capsule.
-polyglutamic acid (
-PGA)
capsule, is encoded on plasmid pXO2 (95 kbp) (1). The capsule is only
weakly immunogenic and is therefore not suitable for vaccine purposes
(2, 3).
-PGA is synthesized by enzymes encoded by the
capA, capB, and capC genes (4). The
-PGA capsule forms the outermost element of the B. anthracis cell where it inhibits phagocytosis (2) by providing
a monotonous linear polymer.
-amylase. Increasing the cellular concentration of PrsA
results in a corresponding increase in the amount of
-amylase
secreted into the culture medium (13). Here we show that secretion of rPA, like that of
-amylase, is PrsA-dependent.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-D thiogalactopyranoside (IPTG) was
added at 1 mM and xylose at 1% (w/v) to induce gene expression from the Pspac and Pxyl promoters,
respectively.
Bacterial strains and plasmids
-Amylase Activity--
-Amylase activity
in culture supernatants was determined using the Phadebas amylase test
(Pharmacia & Upjohn, Kalamazoo, MI).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-amylase derived from Bacillus amyloliquefaciens
(12). The prsA3 mutation appears to affect PrsA folding,
resulting in post-translocational proteolysis of PrsA and reduced PrsA
activity (16). To determine whether PrsA influences the production of
rPA, the pagA-encoding plasmid pYS5 (17) was introduced into
B. subtilis strain MFJ945 in which the wild-type
prsA gene was replaced with prsA3. The isogenic parental strain with a normal copy of prsA was MFJ943.
MFJ945 (prsA3) and MFJ943 (prsA) were grown in
complete anthracis medium and the cultures sampled during growth and
stationary phases. Analysis of the culture supernatants by SDS-PAGE
(Fig. 1) indicated that exponential and
early stationary phase samples of MFJ943 contained a protein with the
same relative mobility as rPA (lanes 2-3). This protein was
absent from overnight cultures of this strain (lane 4). In
contrast, no rPA was detected in the supernatants of MFJ945
(prsA3) at any growth stage (lanes 5-7).
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Fig. 1.
SDS-PAGE analysis of rPA released into
culture supernatants during exponential (5.5 h,
A450 ~ 0.5), early stationary (7 h,
A450 ~ 3.0), and late stationary
phase (21.5 h, A450 ~ 4.0) by
B. subtilis strains encoding wild-type or mutant versions
prsA. Protein size markers (kDa), lane 1; MFJ943
(5.5 h), lane 2; MFJ943 (7 h), lane 3; MFJ943
(21.5 h), lane 4; MFJ945 (5.5 h), lane 5; MFJ945
(7 h), lane 6; MFJ945 (21.5 h), lane 7; PA (1 µg), lane 8; MFJ683 (non-rPA encoding control strain,
21.5 h, lane 9).
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Fig. 2.
Production of rPA by RCW201PA in the presence
of various concentrations of IPTG (0.5-10
mM). Quantitation of Western blot data.
Error bars represent 1 S.E. from the mean of triplicate
experiments.
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Fig. 3.
Multiple alignments of the protein sequences
of B. subtilis PrsA and YacD and B. anthracis PrsAA, PrsAB, and PrsAC. The protein
sequences were aligned using the ClustalW tool
(www.ebi.ac.uk/clustalw). The PpiC PPIase domains I and II
are indicated by the solid and broken boxes,
respectively.
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Fig. 4.
The regions of the B. subtilis and B. anthracis chromosomes encoding
prsA and homologues. Comparative analysis with the
B. subtilis genome (genolist.pasteur.fr/SubtiList) shows
genes with no B. subtilis homologues (speckled);
genes with B. subtilis homologues and showing conservation
of gene order with respect to Bsu-prsA (dark);
genes with B. subtilis homologues and showing conservation
of gene order at a distal location (light); genes with
B. subtilis homologues and showing no conservation of gene
order with B. subtilis (no shading). The
values on the PrsA homologues represent relative identity (%) with
Bsu-PrsA. The values at the start and end of each region are
the coordinates (bp) with respect to the nominal start of
the respective genomes as provided at SubtiList
(genolist.pasteur.fr/SubtiList) and The Comprehensive Microbial
Resource (www.tigr.org).
-amylase) generated strains RCW303, RCW304, and RCW305,
respectively. The resulting strains expressed either the native
B. subtilis prsA gene (+IPTG/
xylose) or the B. anthracis homologue (
IPTG/+xylose) or both (+IPTG/+xylose). As a
control, strain RCW302 was constructed in which a second copy of the
B. subtilis prsA gene was similarly located at the
amyE locus under xylose-inducible control.
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Fig. 5.
Activity of the B. anthracis
prsA orthologues in B. subtilis with respect to
their ability to complement the lethal phenotype of prsA.
All strains were derivatives of RCW201 (Pspac
Bsu-prsA): RCW301 (Pspac Bsu-prsA;
Pxyl), RCW302 (Pspac
Bsu-prsA; Pxyl Bsu-prsA),
RCW303 (Pspac Bsu-prsA;
Pxyl Ban-prsAA), RCW304
(Pspac Bsu-prsA; Pxyl
Ban-prsAB), RCW305 (Pspac Bsu-prsA;
Pxyl Ban-prsAC). Strains were grown
in LB broth with 0.1 mM IPTG, washed, and then enumerated
on LB agar supplemented as follows: unsupplemented (no
shading), 1 mM IPTG (diagonal shading), 1 mM IPTG and 1% xylose (wavy shading), 1%
xylose (horizontal shading). Colonies observed on
unsupplemented LB agar were shown to be IPTG-independent (data not
shown).
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Fig. 6.
The influence on B. subtilis
colony morphology expressing either B. subtilis
or B. anthracis PrsA. Wild-type B. subtilis 168 and complementation mutants RCW302 (Pspac
Bsu-prsA; Pxyl Bsu-prsA),
RCW303 (Pspac Bsu-prsA;
Pxyl Ban-prsAA), RCW304
(Pspac Bsu-prsA; Pxyl
Ban-prsAB), RCW305 (Pspac Bsu-prsA;
Pxyl Ban-prsAC) were grown on LB agar
supplemented with either IPTG (1 mM) or xylose (1%).
-amylase activities in 24-h culture supernatants. RCW101, in which
B. subtilis prsA was expressed from its native promoter, displayed a yield of AmyQ severalfold higher than the strains in which
the prsA genes were under xylose regulation. Of the latter strains, RCW302Amy (with Bsu-prsA) and RCW304Amy (with
Ban-prsAB) exhibited a 2- to 3-fold higher yield of AmyQ
than strains RCW303 (with Ban-prsAA) and RCW305 (with
Ban-prsAC).
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Fig. 7.
Influence of B. subtilis and
B. anthracis PrsA proteins on the yield of
-amylase in culture supernatants. Growth
(solid line) and AmyQ yields (broken line) of
B. subtilis strains expressing B. subtilis and B. anthracis prsA genes.
Bsu-prsA (crosses) was expressed from its native
promoter in RCW101. Bsu-prsA (squares,
RCW302Amy), Ban-prsAA (diamonds, RCW303Amy),
Ban-prsAB (circles, RCW304Amy), and
Ban-prsAC (triangles, RCW305Amy) were expressed
from the xylose-inducible promoter, Pxyl. Strains were
grown in LB broth with 1% xylose.
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Fig. 8.
Influence of B. subtilis and
B. anthracis PrsA proteins on the yield of
-amylase in culture supernatants. Growth
(solid line) and rPA yields (broken line) of
B. subtilis strains expressing B. subtilis and B. anthracis prsA genes.
Bsu-prsA (crosses) was expressed from its native
promoter in RCW 102. Bsu-prsA (squares,
RCW302Amy), Ban-prsAA (diamonds, RCW303PA),
Ban-prsAB (circles, RCW304PA), and
Ban-prsAC (triangles, RCW305PA) were expressed
from the xylose-inducible promoter, Pxyl. Strains were
grown in LB broth supplemented in 1% xylose.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-amylase activities (12). However, because PrsA is
required for B. subtilis viability, it is likely
that at least one protein essential for cell wall synthesis is
PrsA-dependent.
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ACKNOWLEDGEMENT |
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We thank Dr. Jerry Keith for providing laboratory facilities (for M. F. J.).
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FOOTNOTES |
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* This work was supported by grants from the Biotech program of the European Commission (QLK3-CT-1999-00413), the UK Biotechnology and Biological Research Council, and the Defense Science and Technology Laboratory, UK. The work was carried out within the framework of the European Bacillus Secretion Group.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
This paper is dedicated to the memory of Dr. Costa Anagnastopoulos, the father of Bacillus genetics, who died January 2, 2003.
¶ Present address: mjacobs5{at}jhu.edu.
** To whom correspondence should be addressed. Tel.: 44-191-222-7708; Fax: 44-191-222-7736; E-mail: colin.harwood@ncl.ac.uk.
Published, JBC Papers in Press, February 26, 2003, DOI 10.1074/jbc.M301244200
2 R. C. Williams, A. Wipat, and C. R. Harwood, unpublished results.
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ABBREVIATIONS |
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The abbreviations used are:
PA, protective
antigen;
rPA, recombinant PA;
LB, Luria Bertani;
IPTG, isopropyl--D thiogalactopyranoside;
Bsu, B.
subtilis;
Ban, B. anthracis.
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REFERENCES |
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---|
1. | Pezard, C., Berche, P., and Mock, M. (1991) Infect. Immun. 59, 3472-3477[Medline] [Order article via Infotrieve] |
2. | Makino, S., Uchida, I., Terakado, N., Sasakawa, C., and Yoshikawa, M. (1989) J. Bacteriol. 171, 722-730[Medline] [Order article via Infotrieve] |
3. | Mock, M., and Fouet, A. (2001) Annu. Rev. Microbiol. 55, 647-671[CrossRef][Medline] [Order article via Infotrieve] |
4. | Avakyan, A. A., Katz, L. N., Levina, K. N., and Pavlova, I. B. (1965) J. Bacteriol. 90, 1082-1095[Medline] [Order article via Infotrieve] |
5. | Belton, F. C., and Strange, R. E. (1954) Br. J. of Exp. Pathol. 35, 144-152 |
6. | Leppla, S. H. (1991) in Anthrax Toxin Complex; Source Book of Bacterial Protein Toxins (Alouf, J. E. , and Freer, J. H., eds) , pp. 277-301, Academic Press, London |
7. |
Barnard, J. P.,
and Friedlander, A. M.
(1999)
Infect. Immun.
67,
562-567 |
8. | Turnbull, P. C. B. (2000) Curr. Opin. Infect. Dis. 13, 113-120[Medline] [Order article via Infotrieve] |
9. | Ivins, B. E., and Welkos, S. L. (1986) Infect. Immun. 54, 537-542[Medline] [Order article via Infotrieve] |
10. | Wu, X.-C., Wilson, L., Tran, L., and Wong, S.-L. (1991) J. Bacteriol. 173, 4952-4958[Medline] [Order article via Infotrieve] |
11. | Baillie, L. W., Johnson, M., and Manchee, R. J. (1994) Lett. Appl. Microbiol. 19, 225-227[Medline] [Order article via Infotrieve] |
12. | Kontinen, V. P., and Sarvas, M. (1993) Mol. Microbiol. 8, 727-737[Medline] [Order article via Infotrieve] |
13. |
Vitikainen, M.,
Pummi, T.,
Airaksinen, U.,
Wahlstrom, E.,
Wu, H. Y.,
Sarvas, M.,
and Kontinen, V. P.
(2001)
J. Bacteriol.
183,
1881-1890 |
14. | Bron, S. (1990) in Molecular Biological Methods for Bacillus (Harwood, C. R. , and Cutting, S. M., eds) , pp. 75-174, John Wiley and Sons, Chichester, UK |
15. | Vagner, V., Dervyn, E., and Ehrlich, S. D. (1998) Microbiology 144, 3097-3104[Abstract] |
16. |
Hyyrylainen, H. L.,
Vitikainen, M.,
Thwaite, J.,
Wu, H. Y.,
Sarvas, M.,
Harwood, C. R.,
Kontinen, V. P.,
and Stephenson, K.
(2000)
J. Biol. Chem.
275,
26696-26703 |
17. |
Singh, Y.,
Chaudhary, V. K.,
and Leppla, S. H.
(1989)
J. Biol. Chem.
264,
19103-19107 |
18. | Altschul, S. F., Gish, W., Miller, W., Myers, E. W., and Lipman, D. J. (1990) J. Mol. Biol. 215, 403-410[CrossRef][Medline] [Order article via Infotrieve] |
19. |
Pragai, Z.,
and Harwood, C. R.
(2000)
J. Bacteriol.
182,
6819-6823 |
20. | Miller, J., McBride, B. W., Manchee, R. J., Moore, P., and Baillie, L. W. (1998) Lett. Appl. Microbiol. 26, 56-60[CrossRef][Medline] [Order article via Infotrieve] |
21. | Lenz, L. L., and Portnoy, D. A. (2002) Mol. Microbiol. 45, 1043-1056[CrossRef][Medline] [Order article via Infotrieve] |
22. | Bensing, B. A., and Sullam, P. M. (2002) Mol. Microbiol. 44, 1081-1094[CrossRef][Medline] [Order article via Infotrieve] |
23. |
Tjalsma, H.,
Bolhuis, A.,
Jongbloed, J. D. H.,
Bron, S.,
and van Dijl, J. M.
(2000)
Microbiol. Mol. Biol. Rev.
64,
515-547 |
24. | Bron, S., Bolhuis, A., Tjalsma, H., Holsappel, S., Venema, G., and van Dijl, J. M. (1998) J. Biotechnol. 64, 3-13[CrossRef][Medline] [Order article via Infotrieve] |
25. |
Tjalsma, H.,
Noback, M.,
Bron, S.,
Venema, G.,
Yamane, K.,
and van Dijl, J. M.
(1997)
J. Biol. Chem.
272,
25983-25992 |
26. |
Stover, A. G.,
and Driks, A.
(1999)
J. Bacteriol.
181,
1664-1674 |
27. |
Stover, A. G.,
and Driks, A.
(1999)
J. Bacteriol.
181,
5476-5481 |
28. |
Jongbloed, J. D. H.,
Martin, U.,
Antelmann, H.,
Hecker, M.,
Tjalsma, H.,
Venema, G.,
Bron, S.,
van Dijl, J. M.,
and Muller, J.
(2000)
J. Biol. Chem.
275,
41350-41357 |
29. | van Dijl, J. M., Braun, P. G., Robinson, C., Quax, W. J., Antelmann, H., Hecker, M., Muller, J. P., Tjalsma, H., Bron, S., and Jongbloed, J. D. H. (2002) J. Biotechnol. 98, 243-254[CrossRef][Medline] [Order article via Infotrieve] |
30. | Anagnastopoulos, C., and Spizizen, J. (1961) J. Bacteriol. 81, 741-746 |
31. | Palva, I. (1982) Gene 19, 81-87[CrossRef][Medline] [Order article via Infotrieve] |
32. | Lacey, R. W., and Chopra, I. (1976) J. Med. Microbiology 7, 285-297 |