Production of Bacillus anthracis Protective Antigen Is Dependent on the Extracellular Chaperone, PrsA*

Rachel C. WilliamsDagger , Mark L. ReesDagger , Myra F. Jacobs§, Zoltán PrágaiDagger , Joanne E. ThwaiteDagger ||, Leslie W. J. Baillie||, Peter T. EmmersonDagger , and Colin R. HarwoodDagger **

From the Dagger  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

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 gamma -polyglutamic acid capsule.

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 gamma -polyglutamic acid (gamma -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). gamma -PGA is synthesized by enzymes encoded by the capA, capB, and capC genes (4). The gamma -PGA capsule forms the outermost element of the B. anthracis cell where it inhibits phagocytosis (2) by providing a monotonous linear polymer.

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 alpha -amylase. Increasing the cellular concentration of PrsA results in a corresponding increase in the amount of alpha -amylase secreted into the culture medium (13). Here we show that secretion of rPA, like that of alpha -amylase, is PrsA-dependent.

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.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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-beta -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.


                              
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Table I
Bacterial strains and plasmids

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 alpha -Amylase Activity-- alpha -Amylase activity in culture supernatants was determined using the Phadebas amylase test (Pharmacia & Upjohn, Kalamazoo, MI).

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).

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Influence of PrsA on rPA Secretion-- Mutants of prsA encoding a defective PrsA protein exhibit a defect in the secretion of AmyQ, an alpha -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).

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.


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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.

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.


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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.

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.


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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).

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 alpha -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.

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).


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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).

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.


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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%).

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 alpha -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 alpha -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.

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.


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Fig. 8.   Influence of B. subtilis and B. anthracis PrsA proteins on the yield of alpha -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

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 alpha -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.

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

    ACKNOWLEDGEMENT

We thank Dr. Jerry Keith for providing laboratory facilities (for M. F. J.).

    FOOTNOTES

* 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.

    ABBREVIATIONS

The abbreviations used are: PA, protective antigen; rPA, recombinant PA; LB, Luria Bertani; IPTG, isopropyl-beta -D thiogalactopyranoside; Bsu, B. subtilis; Ban, B. anthracis.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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