Selection and characterization of a promoter for expression of single-copy recombinant genes in Gram-positive bacteria
© Provvedi et al; licensee BioMed Central Ltd. 2005
Received: 22 October 2004
Accepted: 14 January 2005
Published: 14 January 2005
In the past ten years there has been a growing interest in engineering Gram-positive bacteria for biotechnological applications, including vaccine delivery and production of recombinant proteins. Usually, bacteria are manipulated using plasmid expression vectors. The major limitation of this approach is due to the fact that recombinant plasmids are often lost from the bacterial culture upon removal of antibiotic selection. We have developed a genetic system based on suicide vectors on conjugative transposons allowing stable integration of recombinant DNA into the chromosome of transformable and non-transformable Gram-positive bacteria.
The aim of this work was to select a strong chromosomal promoter from Streptococcus gordonii to improve this genetic system making it suitable for expression of single-copy recombinant genes. To achieve this task, a promoterless gene encoding a chloramphenicol acetyltransferase (cat), was randomly integrated into the S. gordonii chromosome and transformants were selected for chloramphenicol resistance. Three out of eighteen chloramphenicol resistant transformants selected exhibited 100% stability of the phenotype and only one of them, GP215, carried the cat gene integrated as a single copy. A DNA fragment of 600 base pairs exhibiting promoter activity was isolated from GP215 and sequenced. The 5' end of its corresponding mRNA was determined by primer extention analysis and the putative -10 and a -35 regions were identified. To study the possibility of using this promoter (PP) for single copy heterologous gene expression, we created transcriptional fusions of PP with genes encoding surface recombinant proteins in a vector capable of integrating into the conjugative transposon Tn916. Surface recombinant proteins whose expression was controlled by the PP promoter were detected in Tn916-containing strains of S. gordonii and Bacillus subtilis after single copy chromosomal integration of the recombinant insertion vectors into the resident Tn916. The surface recombinant protein synthesized under the control of PP was also detected in Enterococcus faecalis after conjugal transfer of a recombinant Tn916 containing the transcriptional fusion.
We isolated and characterized a S. gordonii chromosomal promoter. We demonstrated that this promoter can be used to direct expression of heterologous genes in different Gram-positive bacteria, when integrated in a single copy into the chromosome.
In the past ten years there has been a growing interest in engineering Gram-positive bacteria for biotechnological applications, including vaccine delivery. [1–4], and in situ production of anti-infective protectants  and microbicides . A common approach to genetic manipulation of bacteria is based on the use of plasmid expression vectors since these recombinant molecules can be introduced into bacterial cells by a variety of genetic techniques such as natural transformation, artificial transformation, transduction, conjugative mobilization, and electroporation [7–9]. However, the major limitation of this approach is due to the fact that recombinant plasmids are often lost from the bacterial culture upon removal of antibiotic selection. Certainly, this has consequences when using recombinant bacteria in vivo where their replication occurs in the absence of selection. An alternative approach is to integrate recombinant DNA molecules into the bacterial chromosome since this method allows increased in vivo stability of the genetic constructs. Therefore a lot of efforts have focused on the development of efficient expression systems based on chromosomal integration of expression cassettes [10, 11]. Naturally transformable bacteria represent a convenient model, since heterologous DNA can be easily integrated into their chromosomes, whereas genetic manipulation of non-transformable bacteria is more difficult and relies mainly on electroporation and conjugative mobilization of foreign DNA molecules.
We have previously described a genetic system based on conjugative transposons allowing stable integration of recombinant DNA into the chromosome of transformable and non-transformable streptococci [12, 13]. A series of transposon insertion vectors containing two regions of homology with Tn916  have been created in order to manipulate both naturally transformable and non-transformable Gram-positive bacteria carrying Tn916 . The aim of this work was to select a strong promoter to improve this genetic system making it suitable for expression of single-copy recombinant genes in a broad spectrum of Gram-positive bacteria.
Results and discussion
Promoter selection by chromosomal integration
In vivo analysis of promoter strength was determined in the eighteen selected transformants by measuring the minimum inhibitory concentration (MIC) of Cm. Fifteen transformants exhibited a MIC of 16 μg/ml, whereas the remaining three transformants exhibited a MIC of 8 μg/ml. The fifteen strains with higher MIC were tested for the stability of the chloramphenicol-resistance phenotype: after 50 generations of growth without selection, bacterial cultures were plated on non-selective medium and at least 200 colonies were picked and tested for Cm resistance. The stability of the resistance phenotype varied considerably among the different transformants (data not shown). Three strains (GP214, GP215 and GP216) showed a 100% stability and were chosen for further analysis.
Cloning of a promoter from S. gordonii
The structure of the integrated cat gene in GP214, GP215 and GP216 was analyzed by Southern blot. Only GP215 showed to have a single cat copy integrated into the chromosome, whereas in GP214 and GP216 integration occurred at multiple sites (data not shown). In order to clone the regions flanking cat integration site in GP215, the chromosome of this strain was cut with TaqI whose recognition sequence is absent inside the cat gene. The derivative fragments were ligated to pBLUESCRIPT, and the ligation mixture was used to transform Escherichia coli cells; transformants were then selected for CmR. All transformants analyzed for plasmid content showed to carry a plasmid of the same size. One of these transformants (GP334) was selected for further analysis and the transforming plasmid was named pVMB5. By restriction analysis we showed that pVMB5 contains a 2.2 kb TaqI insert where a 600 base pairs (bp) streptococcal DNA fragment was cloned upstream of the cat gene. This DNA fragment was stably maintained in E. coli, where it retained its promoter activity, conferring CmR to E. coli. This is of considerable interest since it is known that very often cloning streptococcal promoters in a high copy number plasmid results in the failure of that plasmid to be established .
Identification of the transcriptional start site
Insertion vectors to express heterologous proteins
Expression of recombinant proteins in Gram-positive bacteria
S. gordonii, B. subtilis, and Enterococcus faecalis were three Gram-positive hosts used to analyze the capability of PP to direct transcription of the heterologous genes expressing M6, M6/TTFC and M6/OVA recombinant proteins.
Expression of M6 in S. gordonii
Expression of M6/TTFC in B. subtilis
Competent cells of B. subtilis GP800.2, containing one copy of Tn916 integrated into the chromosome, were transformed with the insertion vector pSMB288, bearing the transcriptional fusion PP-emm6/ttfc. One transformant, GP848, was chosen for further studies. A culture of GP848 was grown to mid-exponential phase and analyzed by Western-blotting for the presence of recombinant M6/TTFC. As shown in Fig. 6B, two reactive bands could be detected in the envelope fraction. The lower band, indicated by an arrow, corresponds to the mature protein (predicted molecular weight, 82 kDa), while the upper band probably represents an unprocessed form (predicted molecular weight, 86.4 kDa).
Expression of M6/OVA in E. faecalis
We have isolated and characterized a promoter from the chromosome of S. gordonii, and demonstrated that it can be used to direct expression of heterologous genes in different Gram-positive bacteria when integrated in a single copy into the chromosome. This promoter, together with the genetic system based on suicide vectors able to integrate into conjugative transposons, represents a useful tool for the stable manipulation of a broad spectrum of Gram-positive bacteria.
Bacterial strains, plasmids and growth conditions
Strains and plasmids used in this work
Recipient in transformation
ΩTn5253, SmR, CmR, TcR
pKT random in V288, CmR
pKT random in V288, CmR
pKT random in V288, CmR
emm6, ErR, SmR
ΩTn5253 (ΔtetM::pSMB139) ErR, SmR, CmR, TcS
Recipient in conjugation, SmR, SpR
ΩTn916, ErR, TcS (Δ tetM::pSMB156)
ΩTn916, ErR, TcS (Δ tetM::pSMB156)
ΩTn916, ErR, TcS (Δ tetM::pSMB288)
pBluescript::PP-cat, ApR, CmR
pBluescript::emm6.1::ermC, ApR, ErR
pVA891 derivative containing DNA sequences from Tn5253, CmR, ErR
pVA891::emm6, CmR, ErR
pVA891::PP-emm6, CmR, ErR
pSMB47::PP-emm6, CmR, ErR
pSMB47::PP-emm6/55, CmR, ErR
pSMB47::PP-emm6/55::ova, CmR, ErR
Total DNA preparation of S. gordonii was performed as previously described . Plasmid DNA was prepared using the Qiagen Plasmid Kit (Qiagen) according to the manufacturer's instructions. All recombinant techniques were performed following standard procedures , using E. coli HB101 or DH5α as a host. DNA restriction enzymes were obtained from Roche and used according to the manufacturer's instructions.
Promoter selection by chromosomal integration
A 1.6 kb HindIII-BamHI fragment of plasmid pKT , containing a cat gene, was ligated with random chromosomal DNA fragments of S. gordonii V288 previously cut with both HindIII and BamHI. The initiation codon of cat was 34 bp downstream of the HindIII site, therefore HindIII cleavage would leave cat promoterless and preceded by an intact ribosome binding site. The ligation mixture was introduced in S. gordonii V288 and transformants were selected for CmR.
The MIC of chloramphenicol for S. gordonii was determined following standard procedures .
Construction of recombinant vectors
A 1648 bp fragment containing the emm6 gene (encoding M6, a fibrillar surface protein of S. pyogenes) was amplified by PCR from plasmid pVMB20  using the oligonucleotides 5'-ATGGATCCATCATATGGCTAAAAATAACACGAAT-3' (upstream primer, containing a BamHI site and introducing a NdeI site at the ATG translation initiation codon) and 5'-GCATGTCGACCATAATCATTAAATGTATCTCAT-3' (downstream primers containing a SalI site). This 1648 bp PCR fragment was digested with BamHI and SalI and cloned in pVA891  previously digested with BamHIand SalI, resulting in plasmid pSMB89. A 451 bp region containing the streptococcal promoter PP was amplified from pVMB5 using the following primers: 5'-CGAGGATCCTTTAATCGATACTCATG-3' (upstream primer, containing a BamHI and a ClaI site) and 5'-CCGCATATGGTTCTCCTTTTTATTTGT-3' (downstream primers containing a NdeI site). After digestion with BamHI and NdeI, this PCR product was inserted between the BamHI and NdeI site of pSMB89 to obtain a transcriptional fusion of PP with emm6. The resulting plasmid was named pSMB128. This plasmid was first cut with BamHI, treated with Klenow enzyme to generate blunt ends, and finally cut with SalI to obtain a 2.0 kbfragment containing PP-emm6 fusion. This fragment was gel-purified and ligated to the suicide integrative plasmid pSMB47  previously cut with HindIII, treated with Klenow enzyme to generate blunt ends, and finally cut with SalI. The resulting plasmid was named pSMB139 (Fig. 4). To introduce a multiple cloning site in the emm6 gene contained in pSMB139, the 900 bp AvrII-HindIII fragment internal to emm6 was replaced with the 390 bp AvrII-HindIII fragment of emm6 from pSMB55  (Fig. 4). The resulting plasmid was named pSMB148. A 1016 bp DNA region encoding 339 aa of the chicken ovalbumin (OVA) (Gene Bank accession number: V00383) was amplified with the following primers: 5'-CTAGATCTGACAGCA CCAGGACAC-3' (upstream primer containing a BglII site) and 5'-TAAAGCTTTAGGGG AAACACATCTG-3' (downstream primer containing a HindIII site). After digestion with BglII and HindIII, this segment was introduced in pSMB148 previously digested with BglII and HindIII. The resulting plasmid, named pSMB156, contained a translational fusion of M6 with OVA. To create a translational fusion of M6 with the tetanus toxin fragment C (TTFC) a 1374 bp BglII-HindIII fragment encoding 458 aa of TTFC was isolated from pSMB158  and cloned between the BglII and HindIII sites of pSMB148. The resulting plasmid was named pSMB288.
Preparation of S. gordonii and B. subtilis cell envelope fractions (representing the protoplast surface containing the cell membrane together with cell wall fragments associated to the protoplasts) was performed as already described [29, 30]. The monoclonal antibody 10B6  diluted 1:1000 was used to detect the presence of M6 protein. M6/TTFC fusion protein was visualized with an anti-TTFC rabbit serum (Calbiochem-Novabiochem Corporation) diluted 1:1000.
DNA sequence determination
Total RNA was isolated from a 50 ml cell culture of S. gordonii and E. coli grown to late exponential phase (OD590 ≌ 0.5). Cells were harvested by centrifugation at 6000 × g at 4°C and lysed according to the following procedures. E. coli cells were first resuspended in hot (100°C) lysis buffer (50 mM Tris/HCl pH8, 1 mM EDTA, 1% SDS) and lysed by boiling the suspension for 5 minutes. S. gordonii cells were resuspended in lysozyme buffer (25 mM Tris/HCl (pH8), 10 mM EDTA, 50 mM glucose) and subjected to three cycles of freezing in liquid nitrogen and thawing at 52°C. After incubating with 0.2 mg/ml of lysozyme for 30 min at 37°C, one volume of hot (100°C) lysis buffer (100 mM Tris/HCl (pH8), 2 mM EDTA, 2% SDS) was added, and complete lysis was obtained by boiling cells for 5 minutes. After boiling, all lysates were cooled on ice for 5 min and total RNA was purified using the SV Total RNA Isolation System (Promega).
Primer extention analysis was performed with a synthetic oligonucleotide 5'-GTTCTTTACGATGCC-3' (position 47 to 61 relative to the initiation of the cat gene). Two pmol of the oligonucleotide, labeled with [γ-32P] ATP (3000 Ci/mmol, Amersham) using T4 polynucleotide kinase (New England Biolabs), were precipitated with 10 μg of RNA and the pellet was resuspended in 8 μl of Moloney Murine Leukemia Virus (M-MuLV) Reverse Transcriptase buffer (50 mM Tris/HCl (pH8.3), 8 mM MgCl2, 10 mM DTT). The mixture was heated at 65°C for 3 min, cooled rapidly at -80°C for 1 min and then transfered on ice until it was completely thawed. 1 μl of a 3.75 mM deoxynucleoside triphosphate solution and 10 U of M-MuLV Reverse Transcriptase (New England Biolabs) were added to the RNA-primer hybrid. The reaction mixture was incubated at 48°C for 30 min and terminated with 10 ml of stop solution (95% formamide, 20 mM EDTA pH8.0, 0.05% bromophenol blue, and 0.05% xylene cyanol FF). The reverse transcriptase reactions were analyzed by electrophoresis on a 6% polyacrylamide-7 M urea gel with sequencing reaction obtained with the same primer used as size standards.
Conjugation experiments were performed on solid media as previously described .
The work was supported in part by the European Commission grant QLK2-CT2000-00543 to GP, a grant from MIUR (COFIN 2002) to GP and from MIUR (FIRB RBAU01X9TB) to MRO.
The authors wish to thank Claudio Gualerzi for help and advice with the primer extention analysis.
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