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Journal of Bacteriology, November 2001, p. 6294-6301, Vol. 183, No. 21
Dipartimento di Fisiologia Generale ed
Ambientale, Sezione di Microbiologia, Università Federico II,
Naples,1 and Laboratorio di
Microbiologia Molecolare e Biotecnologia, Sezione di Microbiologia,
Dipartimento di Biologia Molecolare, Università di Siena,
Siena,2 Italy
Received 16 April 2001/Accepted 8 August 2001
We developed a novel surface display system based on the use of
bacterial spores. A protein of the Bacillus subtilis spore coat, CotB, was found to be located on the spore surface and used as
fusion partner to express the 459-amino-acid C-terminal fragment of the
tetanus toxin (TTFC). Western, dot blot and fluorescent-activated cell
sorting analyses were used to monitor TTFC surface expression on
purified spores. We estimated that more than 1.5 × 103 TTFC molecules were exposed on the surface of each
spore and recognized by TTFC-specific antibodies. The efficient surface presentation of the heterologous protein, together with the simple purification procedure and the high stability and safety record of
B. subtilis spores, makes this spore-based display system a potentially powerful approach for surface expression of bioactive molecules.
Presentation of heterologous
proteins in a biologically active form is an important task with
potential applications (11, 32) in a variety of fields
ranging from live-vaccine development (19-21, 24) to
treatment of microbial infections (3), peptide library
screening (4), and biocatalyst or bioadsorbent development (25, 31).
Several approaches have been undertaken to develop efficient display
systems expressing heterologous polypeptides on the surface of cells
(4, 11, 29, 32) and viruses (2, 7, 26, 30).
In gram-negative bacteria, various surface proteins such as outer
membrane proteins LamB and OmpA and lipoproteins have been exploited as
fusion partners to express bacterial and viral antigens and induce
specific antibody response in animals immunized with the recombinant
bacteria (19, 29).
Several gram-positive bacteria have also been used for the expression
of heterologous proteins, mainly antigenic determinants for the
induction of both local and systemic antibody response in animal models
(3, 20, 21, 24). In addition, many gram-positive species
have been considered for purposes other than vaccine development. Because of the thicker cell wall, gram-positive bacteria are viewed as
preferential candidates over gram-negative bacteria for the development
of bacterial biocatalysts and whole-cell adsorbents (19,
29).
Here we report a novel surface display system based on the use of
bacterial spores. There are many potential advantages with the
utilization of spores: (i) a high stability of the expression system is
ensured by the well-documented resistance and stability of the
bacterial spore (9, 14); (ii) a good safety record is
ensured by the use of spores of species including Bacillus subtilis, B. clausii, B. coagulans, B. cereus, and B. natto as food additives in human and
animal food preparations and as prescription or nonprescription
products for the treatment of gastrointestinal disorders (12,
15); (iii) simple and economic production of large amounts of
spores is ensured by already available and commonly used procedures for
industrial-scale production and commercialization of several
spore-based products (12, 15).
Among the various Bacillus species, B. subtilis
offers additional advantages due to the detailed knowledge of its spore
structure (9, 14) and the availability and ease of
advanced genetic tools (5) and genomic data
(17) that facilitate the construction of recombinant
spores. B. subtilis spores are surrounded by a coat, a
proteinaceous structure organized into two layers and composed of at
least 20 polypeptides (9, 14). Some of these, like CotA,
CotB, CotC (8), CotF (6), and CotG
(27), have been associated with the outer part of the coat
and are referred to as outer coat proteins (9, 14), but
for all of them the exact location within the coat and the protein
domain required for external localization are not known.
Here we show that the previously identified outer coat component CotB
(8) (Fig. 1A) is localized
on the spore surface, and based on this, we report the use of CotB as a
fusion partner for surface display on B. subtilis spores. As
a model system, we expressed the 459-amino-acid C-terminal fragment of
the tetanus toxin (TTFC) (13). This work provides the
first evidence that a heterologous protein can be expressed on the
surface of a bacterial spore and points to this peculiar cell form as a
novel and potentially powerful system to display bioactive molecules.
0021-9193/01/$04.00+0 DOI: 10.1128/JB.183.21.6294-6301.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Surface Display of Recombinant Proteins on Bacillus
subtilis Spores
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
View larger version (27K):
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FIG. 1.
(A) CotB amino acid sequence (16; accession number
P07789). The three 27-amino-acids repeats are underlined. The arrow
indicates the last CotB amino acid residue (at position 275) in fusion
A and fusion B (see below); the 41 amino acid residues used in fusion C
are double underlined (see below). (B) cotB gene and
strategy for construction of CotB-TTFC gene fusions. tetC
(1,137 bp) was cloned in frame to either the 3' (fusions A) or the 5'
(fusion B) end of the 825-bp DNA fragment coding for the first 275 amino acids of CotB. Fusion C was derived from fusion A by cloning of a
DNA fragment (123 bp) coding for the last 41 amino acids of CotB
(double underlined in panel A) in frame with the 3' end of TTFC.
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MATERIALS AND METHODS |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Bacterial strains and transformation.
B. subtilis
strains utilized are listed in Table 1.
Plasmid amplification for nucleotide sequencing, subcloning
experiments, and transformation of B. subtilis competent
cells were performed with Escherichia coli strain DH5
(28). Bacterial strains were transformed by previously
described procedures: CaCl2-mediated transformation of
E. coli competent cells (28) and two-step transformation of B. subtilis (5).
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Construction of gene fusions.
The general strategy for the
construction of the gene fusions is shown in Fig. 1B. Fragments of
cotB DNA were PCR amplified from the B. subtilis
chromosome priming the amplification with the synthetic
oligonucleotides shown in Fig. 1B and listed in Table
2. The PCR products were visualized on
ethidium bromide-stained agarose gels and gel purified by the QIAquick
gel extraction kit (Qiagen) as specified by the manufacturer.
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(i) Fusion A. A purified 1,071-bp DNA fragment originating from the amplification of B. subtilis chromosomal DNA with B1 and B3 oligonucleotides (Table 2) was sequentially digested with SphI and BglII and cloned in frame to the 5' end of the tetC gene carried by plasmid pGEM-TTFC (24), yielding plasmid pNS4.
(ii) Fusion B. A purified 246-bp DNA fragment originating from the amplification of B. subtilis chromosomal DNA with B1 and B6 oligonucleotides (Table 2) was digested with SphI and BamHI and cloned into plasmid pGEM-TTFC (20) in frame with the 5' end of the tetC gene. The plasmid thus obtained was cleaved with BglII and XhoI and used to clone to the 3' end of tetC a second PCR fragment of 825 bp, originating from amplification of the B. subtilis chromosome with B7 and B3 oligonucleotides (Table 2). The resulting plasmid was named pNS16.
(iii) Fusion C. A purified DNA fragment of 123 bp originating from the amplification of B. subtilis chromosomal DNA with B4 and B5 oligonucleotides (Table 2) was digested with SalI and BglII and ligated into plasmid pNS4 (fusion A), yielding plasmid pNS8.
On digestion with enzymes SphI and SalI, pNS4 and pNS16 released a 2,474-bp DNA fragment while pNS9 released a 2,597-bp fragment, all containing the cotB gene region fused to tetC. These fragments were gel purified as described above and ligated into plasmid pDG364 (5) previously digested with the same two enzymes. E. coli competent cells were transformed with the ligation mixture, and the selected ampicillin-resistant clones were screened by restriction analysis of their plasmids. Individual clones for each transformation were selected, named pNS6, pNS17, and pNS9, from pNS4, pNS16, and pNS8, respectively, and used to determine the nucleotide sequence of the inserted DNA.Chromosomal integration. Plasmids pNS6, pNS9, and pNS17 were linearized by digestion with PstI and used to transform competent cells of the B. subtilis strain PY79. Cmr clones were the result of double-crossover recombination, resulting in the interruption of the nonessential amyE gene on the B. subtilis chromosome. All Cmr clones were tested by PCR using chromosomal DNA as a template and oligonucleotides AmyS (5'-CCAATGAGGTTAAGAGTATTCC-3', annealing +569/+590 of amyE) and AmyA (5'-CGAGAAGCTATCACCGCCCAGC-3', annealing +2128/+2150 of amyE) to prime DNA amplification. Clones derived from plasmids pNS6 and pNS17 showed an amplification product of 4,057 bp, while clones derived from plasmid pNS9 showed a slightly larger amplification product (4,180 bp) due to the presence of an additional part of cotB DNA (Fig. 1B), thus indicating the occurrence of correct recombination events. Three clones, one for each transformation, were named RH103 (from pNS6, fusion A in Fig. 1B), RH110 (from pNS17, fusion B in Fig. 1B), and RH109 (from pNS9, fusion C in Fig. 1B) and kept for further studies.
All three fusions were moved into a cotB null mutant strain by chromosomal DNA-mediated transformation (5). Chromosomal DNA extracted from strains RH103, RH109, and RH110 was used to transform the isogenic cotB null strain RH201. RH201 was obtained by transforming strain PY79 with plasmid pRH30, a pUC19 derivative carrying the first 404 bp of the cotB coding region and a spectomycin resistance gene cassette (Spr). Spr clones were the result of a Campbell-like recombination event interrupting cotB. Several Spr clones were analyzed by PCR, and one of them, RH201, used for further studies.Western and dot blot analyses. Sporulation of wild-type and recombinant strains was induced by the exhaustion method (22). After a 24-h incubation at 37°C, spores were collected, washed several times, and purified by lysozyme treatment as previously described (22). The number of purified spores obtained was measured by direct counting with a Bürker chamber under an optical microscope (Olympus BH-2 with 40× lenses).
Aliquots of 1010 spores suspended in 0.30 ml of distilled water were used to extract coat proteins by sodium dodecyl sulfate (SDS)-dithiothreitol (DTT) treatment at 65°C, as previously reported (22). An average of 320 µg (± 2%) of total coat proteins was reproducibly extracted from each aliquot, as estimated by two independent methods: the bicin choninic acid protein assay (Pierce) and the Bio-Rad protein assay. The amount of extracted coat proteins was not dependent on the strain used and corresponded to 0.032 pg of total proteins from each spore. Extracted proteins were fractionated on 10 or 12.5% denaturing polyacrylamide gels, electrotransferred to nitrocellulose filters (Bio-Rad), and used for Western blot analysis by standard procedures. Western blot filters were visualised by the enhanced chemiluminescence (Amersham Pharmacia Biotech) method as specified by the manufacturer. Serial dilutions of extracted proteins and of purified TTFC were used for dot blot analysis. The filters were then visualized by BCIP/NBT color development solution (Bio-Rad) staining and subjected to densitometric analysis with Fluor-S Multimager (Bio-Rad). CotB-specific antibodies were raised in rabbits immunized with a 15-amino-acid synthetic peptide designed on the base of the N-terminal region of CotB (IGtech, Salerno, Italy).Flow cytometry. A total of 105 purified spores were washed in phosphate-buffered saline (PBS) and incubated for 1 h at 37°C with specific antibodies diluted in PBS-1% bovine serum albumin. After four washes in PBS, fluorescein isothiscyanate (FITC)-conjugated anti-mouse immunoglobulin G (IgG) (1:64; Sigma) was added and the mixture was incubated for 1 h at room temperature. Samples were then resuspended in 1 ml of PBS buffer and analyzed using a FACScan instrument equipped with Lysis II software (Becton Dickinson, Mountain View, Calif.) as previously reported (24).
Immunization of mice, sample collection, and analysis. Female BALB/c mice (6 weeks old) were obtained from Charles River (Lecco, Italy) and maintained in our animal facilities for the duration of the experiment. All animal procedures were in accordance with institutional guidelines.
Spores were prepared as previously reported (22) and counted as described above. Two groups of six mice each were inoculated with a single subcutaneous inoculum of 1010 spores from strain RH103, carrying fusion A, and the wild-type isogenic strain. Serum samples were collected from the retro-orbital plexus of anaesthetized mice at weeks 0 and 4 and stored at
70°C until used.
The presence of TTFC-specific serum IgG antibodies was assayed by
enzyme-linked immunosorbent assay as previously described (24). Flat-bottom microtiter plates (high-binding
capacity; Greiner, Frickenhausen, Germany) were coated with 100 µl of
recombinant TTFC per well (1 µg/ml; Boehringer Mannheim), and serum
samples initially diluted 1:20 and titrated in twofold dilutions in
duplicate were added. After a 2-h incubation at 37°C, the plates were
washed and alkaline phosphatase-conjugate goat anti-mouse IgG (1:1,500, Southern Biotechnology Associates, Birmingham, Ala.) was added. The
alkaline phosphatase substrate was added, and the plates were read
after 1 h at 405 nm by using a 340 ATC reader (SLT
Labinstrument). The TTFC-specific IgG concentration in each
sample was calculated using a standard curve of anti-TTFC monoclonal
antibody (Boehringer GmbH, Mannheim, Germany), starting from a
concentration of 8 ng/ml and titrated in twofold dilutions in duplicate.
Samples were tested individually, and data were expressed as the
mean ± standard error of the mean (SEM). Statistical significance was determined by Student's unpaired t test, and the
significance level was set at P 
0.05.
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RESULTS |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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CotB is exposed on the surface of B. subtilis spores. At least 20 polypeptides are organized to form the two layers of the B. subtilis spore coat (9, 14). Some of these polypeptides have been associated with the outer part of the coat, but for all of them the exact location within the coat and the protein domain required for surface exposure are not known. To identify an externally exposed coat component, we investigated the localization of CotB, a 42.9-kDa component of the B. subtilis spore coat associated with the outer coat layer (34) and characterized by a strongly hydrophilic region at the C-terminal end (17). More than 50% of CotB amino acid residues from residues 248 to 356 are serines (Fig. 1A), and three 27-amino-acid repeats are present from residues 253 to 333 (underlined in Fig. 1A).
Spores were purified from a B. subtilis wild-type strain (PY79) (33) and an isogenic mutant strain carrying a Cmr gene cassette (cat) interrupting the cotB coding region (BD067) (8). Purified spores were reacted with CotB-specific antibodies and run in a cytofluorimetric assay (Fig. 2). Specific fluorescent staining of the spore surface was observed only with PY79 spores (Fig. 2), indicating that in wild-type spores CotB was accessible to antibodies and thus probably externally exposed. Based on these results, CotB was selected as a fusion partner to attempt the expression of heterologous polypeptides on the spore surface.
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Construction and chromosomal integration of the CotB-TTFC gene fusion. The strategy to obtain recombinant B. subtilis spores expressing TTFC on their surface was based on (i) use of the cotB gene and of its promoter for the construction of translational fusions and (ii) chromosomal integration of the cotB-tetC gene fusions into the coding sequence of the nonessential gene amyE (5).
To avoid potential stability problems of the genetic constructs, cotB DNA encoding the three 27-amino-acid repeats of CotB (Fig. 1A) was not included in the gene fusions and only DNA encoding the N-terminal 275 amino acids of CotB was used (Fig. 1B). Fusion A (Fig. 1B) was obtained by cloning tetC in frame with the serine codon at position 275 (Fig. 1A); fusion B (Fig. 1B) was constructed by inserting tetC between cotB transcriptional-translational signals and cotB DNA coding for the first 275 amino acids; fusion C (Fig. 1B) was derived from fusion A by addition of DNA coding for the C-terminal 41 amino acids of CotB (Fig. 1A). All gene fusions were integrated on the B. subtilis chromosome at the amyE locus by double-crossover recombination events (see Materials and Methods). Individual clones for each transformation were tested by PCR (data not shown); these clones were named RH103 (fusion A), RH110 (fusion B), and RH109 (fusion C) and were used for further analysis. The three recombinant strains and their isogenic parental strain PY79 showed comparable sporulation and germination efficiencies, and their spores were equally resistant to chloroform and lysozyme treatment (data not shown), indicating that the presence of the CotB-TTFC fusion does not significantly affect spore structure and/or function.CotB-TTFC gene fusions are localized to the spore coat.
To
verify that CotB-TTFC gene fusions were localized to the spore coat, we
analyzed cell extracts of sporulating cells in Western blot experiments
with anti-CotB and anti-TTFC antibodies. Strains RH103 (fusion A),
RH110 (fusion B), and RH109 (fusion C) were grown in Difco sporulation
(DS) medium and harvested 7 h after the onset of
sporulation, at the time of maximal cotB expression
(9, 14). Cells were disrupted by sonication, and the
total-cell extract (supernatant) was separated from forming spores,
unbroken cells, and cell debris (pellet) by centrifugation. The
obtained pellet was then used to extract coat proteins by SDS-DTT
treatment at 65°C. Then 50-µg portions of total proteins obtained
from supernatant and pellet of each strain were fractionated on a 10%
polyacrylamide gel, blotted, and reacted with anti-CotB and anti-TTFC
antibodies. As shown in Fig. 3 (lanes 1 to 8), specific CotB-TTFC signals were observed only in the pellet
fractions of the three strains. Our interpretation of these results was
that CotB-TTFC was assembled on the forming spore (pellet) and, as a
consequence, not found in the cell extracts (supernatant). To determine
whether this interpretation was correct, we used chromosomal DNA-mediated transformation to move the gene fusions into an isogenic B. subtilis strain with the cotG gene deleted
(ER203; Table 1), since CotG is known to be required for CotB assembly
within the spore coat (27). We then repeated the
experiment as described above and collected sporulating cells 7 h
after the onset of sporulation. As shown in Fig. 3 (lanes 9 to 16), in
a cotG null background CotB-TTFC specific signals were found
in the cell extract fraction (supernatant), thus allowing us to infer
that CotB-TTFC gene fusions are spore coat associated.
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Surface display of TTFC on the recombinant spores.
Western
blot analysis of spore coat proteins purified from recombinant strains
revealed the presence of a ca. 82-kDa band which reacted with both
TTFC- and CotB-specific antibodies (Fig. 4A). A 59-kDa band only reacting with
CotB-specific antibodies was present in extracts from wild type and
recombinant spores (Fig. 4A), indicating the presence of intact CotB in
the spore coat, together with the CotB-TTFC fusion protein. To analyze
whether the intact copy of CotB was needed for surface expression of
CotB-TTFC, we used chromosomal DNA-mediated transformation
(5) to move the gene fusions into a cotB mutant
strain (RH201; Table 1). Western blot experiments, performed with coat
proteins from strains carrying the gene fusions in the absence of an
intact copy of cotB, did not show TTFC-specific bands (Fig.
4B), thus indicating that the cotB gene product is needed to
express the CotB-based fusion protein on the spore surface.
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Spore-exposed TTFC is in a biologically active form. Since TTFC immunogenicity is well characterized in mouse models (20), we decided to analyze the TTFC-specific immunogenicity due to the recombinant spores as a measure of the biological activity maintained by TTFC on exposure on the spore. Spores of strain RH103 (carrying fusion A) were purified as previously reported (21) and aliquots of 1010 spores were used to immunize groups of BALB/c mice. After a single subcutaneous inoculum, high levels of TTFC-specific IgG (P = 0.012) were observed in the sera of immunized mice 4 weeks after the inoculum, with a mean ± SEM of 8280 ± 2181 ng/ml, compared to control mice immunized with wild-type spores, which had a mean ± SEM of 4.72 ± 0.71 ng/ml.
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DISCUSSION |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Some features of spore-forming bacilli, such as fast growth, relatively simple nutritional requirements, and efficient secretion of large amounts of proteins and other metabolites, make these organisms particularly suitable for industrial fermentations. Strains of species like B. licheniformis and B. amyloliquefaciens have been used for the production of various proteins of homologous origin, such as proteases, lipases, and starch-degrading enzymes. Some species, especially B. subtilis, have also been used as hosts for the production of heterologous proteins (1, 16, 18). Genetic tools for efficient expression and secretion of recombinant proteins in the latter organism have been rapidly developed and successfully used (10, 23).
In contrast to these extensive applications of vegetative cells of spore-forming bacilli, it is surprising that little attention has been devoted to the spore as a vehicle of recombinant proteins, since the high stability of the spore provides obvious additional advantages. The study reported here is to our knowledge the first to describe the engineering of a spore and to suggest that recombinant B. subtilis spores with relevant features such as safety, stability, easy preparation, and low cost may become very attractive vectors of biologically active molecules.
Detailed genetic and morphological studies have shown that the B. subtilis spore is surrounded by a multilayered coat (9, 14), whose proteinaceous nature immediately suggests the potential possibility of using its structural components as fusion partners for the expression of heterologous proteins on the spore surface. In this study we analyzed this possibility by using as fusion partner CotB, a 380-amino-acid coat protein with a strongly hydrophilic C-terminal end containing three 27-amino-acid repeats (residues 252 to 333) (17). We show here that CotB is externally exposed and can be successfully used to express and expose on the spore surface the highly immunogenic TTFC.
The low fluorescence levels observed when the recombinant spores were reacted with specific anti-TTFC antibodies (Fig. 5) were probably due to the presence on the spore surface of CotB together with CotB-TTFC (Fig. 4). The observed fluorescence was, however, highly specific (no background was obtained with wild-type spores) (Fig. 5D) and very unlikely to be due to broken or defective spores, since sample alterations in cytofluorimetric assays usually result in the appearance of diverse cell types that were not observed in our ungated analysis and since comparable numbers of fluorescent spores were constantly detected in several experiments with all three recombinant strains.
To avoid potential structural instability of the genetic constructs, the three 27-amino-acid repeats of CotB were not included in the fusions with TTFC. The CotB-TTFC coding DNA was put under cotB transcriptional and translational signals and integrated onto the B. subtilis chromosome to ensure correct timing of expression during sporulation and high stability of the genetic construct, respectively.
Unaltered resistance of recombinant spores to chloroform and lysozyme suggested that the presence of TTFC does not affect the structure and/or function of the spore coat. This is not surprising, since most structural coat proteins, including CotA, CotB, CotC, CotD, and CotF, are functionally redundant and the absence of any one of them does not cause evident phenotypic alterations (9, 14).
Surface display of the CotB-TTFC recombinant proteins on the spore appeared similarly efficient for all three fusions, with estimated yields of 1.5 × 103 CotB-TTFC molecules per spore, and was dependent on the presence of an intact copy of CotB. Such dependence is an interesting result that points to a possible interaction among CotB molecules within the coat and that deserves to be specifically addressed in a separate context.
Immunogenicity of the recombinant spores in mice suggested that TTFC exposed on the spore surface is in a biologically active form. Although for ethical reasons we performed the immunization experiments with spores of only one recombinant strain (RH103), the identical results obtained with the all three fusions in Western, dot blot, and cytofluorimetry analyses suggest that TTFC exposed on the surface of spores of the other two strains would be similarly immunogenic in mice.
The size of the heterologous protein to be expressed is often a critical point of surface display systems. The high levels of expression we observed with TTFC, a large peptide of 459 amino acids and a deduced molecular mass of 51.8 kDa, suggest that our spore-based display strategy may have less size restrictions than other cell- or phage-based systems.
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ACKNOWLEDGMENTS |
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This work was supported by European Union grant BIO4 CT960439 to E. Ricca and G. Pozzi and by CNR Targeted Project "Biotecnologie" to E. Ricca and M. De Felice.
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FOOTNOTES |
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* Corresponding author. Mailing address: Dipartimento di Fisiologia Generale ed Ambientale, Università Federico II, via Mezzocannone 16, 80134 Naples, Italy. Phone: 39-081-2534636. Fax: 39-081-5514437. E-mail: ericca{at}unina.it.
Present address: Public Health Research Institute, New York, N.Y.
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Materials and Methods
Results
Discussion
References
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Journal of Bacteriology, November 2001, p. 6294-6301, Vol. 183, No. 21
0021-9193/01/$04.00+0 DOI: 10.1128/JB.183.21.6294-6301.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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