Background 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.
Background
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.Results 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 Tn 916 . 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 Tn 916 . The surface recombinant protein synthesized under the control of PP was also detected in  Enterococcus faecalis  after conjugal transfer of a recombinant Tn 916  containing the transcriptional fusion.
Results
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 Tn 916 . 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 Tn 916 . The surface recombinant protein synthesized under the control of PP was also detected in  Enterococcus faecalis  after conjugal transfer of a recombinant Tn 916  containing the transcriptional fusion.Streptococcus gordoniicatS. gordoniicat916S. gordoniiBacillus subtilis916Enterococcus faecalis916Conclusion 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.
Conclusion
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.S. gordonii

Background 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 [ 5 ] and microbicides [ 6 ]. 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 Tn 916  [ 14 ] have been created in order to manipulate both naturally transformable and non-transformable Gram-positive bacteria carrying Tn 916  [ 12 ]. 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.
Background
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 [ 5 ] and microbicides [ 6 ]. 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.in situin vivoin vivoWe 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 Tn 916  [ 14 ] have been created in order to manipulate both naturally transformable and non-transformable Gram-positive bacteria carrying Tn 916  [ 12 ]. 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.916916Results and discussion Promoter selection by chromosomal integration To select resident promoters from the genome of  Streptococcus gordonii , we performed a random ligation of streptococcal DNA to a promoterless  cat  gene, conferring resistance to chloramphenicol (Cm). The ligation mixture was used to transform the naturally transformable  S. gordonii  «Challis» strain V288 and transformants were selected for Cm resistance. Chromosomal DNA flanking the promoterless  cat  gene provided the homology for the random integration of  cat  into the chromosome during transformation (Fig.  1 ). 71 Cm-resistant (Cm R ) transformants were isolated, presumably as a result of transcriptional fusions of streptococcal promoters to the promoterless  cat  gene. Eighteen Cm R  transformants were selected for further characterization. The strategy commonly used to select promoters is based on cloning random chromosomal DNA fragments in a promoter probe vector upstream of a promoterless reporter gene. However, integrating the promoterless reporter gene ( cat ) directly into the streptococcal chromosome, allowed us to select resident chromosomal promoters expressing  cat  after  in vivo  transcriptional fusion at a single locus on the chromosome. This is preferable when looking for promoters to express heterologous genes integrated into the chromosome in a single copy. 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  Taq I 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 Cm R . 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  Taq I 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 Cm R  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 [ 15 ]. Sequence analysis The streptococcal DNA upstream of the  cat  gene in pVMB5 was sequenced (GenBank accession number: U74080). Analysis of the sequence revealed the presence of an open reading frame (ORF1), preceded by a typical ribosome binding site (RBS) (Fig.  2 ). A BLAST search with the partial sequenced genome of  S. gordonii   showed that ORF1 represents the 5'-end of a gene encoding the first 48 amino acids (aa) of an uncharacterized protein conserved in bacteria and whose function is unknown. As indicated in figure  2 , this truncated streptococcal protein is translationally fused to the N-terminus of CAT. Sequence analysis also revealed that the first 122 bp of the cloned streptococcal fragment belong to the 3'-end of a gene encoding a putative acetyltransferase (ORF2). To obtain information about the chromosomal region containing the 600 bp streptococcal DNA fragment cloned in pVMB5, we looked throughout the whole contig sequence and we found out that upstream ORF2, and partially overlapping with it, there is an ORF encoding a DltD horthologue ( dltD ), a protein involved in D-alanine incorporation into lipoteichoic acid (LTA) [ 16 ], whereas downstream ORF1, there is a gene encoding a Tmp7 transmembrane protein (Fig.  2 ). Identification of the transcriptional start site To identify the sequences responsible for the observed promoter activity, the 5'-end of the  cat- specific mRNA was mapped by primer extension with a specific primer. Total RNA was isolated from  E. coli  GP334 (containing pVMB5) and  S. gordonii  GP215. In both strains, the position of the 5'-end of the mRNA was located at the same purine residue at position 423 of the 600 bp region of streptococcal DNA, 26 nucleotides upstream of the ORF1 translational start site (Fig.  2  and  3 ). Putative -35 and -10 sequences closely resembling the consensus  E. coli  σ 70  and  Bacillus subtilis  σ 43  [ 17 ] recognition sequences TTGACA (-35) and TATAAT (-10) could be identified. The -35 region was TTGCAA, and the -10 region was TAGAAT. The spacing between the -35 and -10 region was 17 bp and was thus similar to the spacing in  B. subtilis  (17 to 19 bp) and  E. coli  (16 to 18 bp) promoters. Moreover, a TG nucleotide pair was found 1 bp upstream of the -10 region; such a structure is typical of Gram-positive bacteria promoters [ 18 ]. Based on this information, we concluded that indeed we isolated a streptococcal promoter which was designated PP. The spacing between the 5'-end of the mRNA and the -10 hexanucleotide was only 2 bp, which is unusually short. A second putative -10 region could be identified in the TATGAT hexanucleotide (Fig.  2 ), whose distance from the 5' end of the mRNA is 7 bp. However, since this sequence is not preceded by the TG nucleotide pair typical of Gram-positive promoters, and is separated from the -35 region only by 12 bp, we suppose that this -10 region of PP is probably not active. Insertion vectors to express heterologous proteins We have previously developed a genetic system based on the use of  Streptococcus pyogenes  surface fibrillar M6 protein, as a partner for the construction of translational fusions to deliver foreign proteins on the surface of  S. gordonii  [ 10 ]. To determine the possibility of using PP for chromosomal single copy heterologous gene expression in Gram-positive bacteria, we first generated a transcriptional fusion of PP with a promoterless gene encoding the M6 protein ( emm6 ) in pSMB47, a suicide vector capable of integrating heterologous DNA into the conjugative transposon Tn 916  via homologous recombinantion [ 12 ]. The recombinant plasmid was named pSMB139 (see Methods) (Fig.  4 ). To construct a Tn 916  insertion vector that could be used to express translational fusions between M6 and any heterologous proteins under the PP promoter control, a 900 bp  Avr II- Hind III fragment internal to  emm6  in pSMB139 was replaced by a 390 bp  Avr II- Hind III fragment of pSMB55 containing a multiple cloning site [ 10 ] (Fig.  4 ). The resulting vector, named pSMB148 (Fig.  4 ), allows to create translational fusions of heterologous proteins between the first 122 N-terminal aa and the last 140 aa of the M6 protein, which provides sequences necessary for cell wall anchoring. pSMB148 was used to create two derivative vectors in which the  emm6  gene was fused respectively with a DNA sequence encoding 339 aa of the chicken ovalbumin (OVA) (pSMB156), and a DNA sequence encoding 458 aa of the tetanus toxin fragment C (TTFC) (pSMB288) (see Methods). A schematic representation of the recombinant proteins is shown in Fig.  5A . 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 pSMB139, bearing a transcriptional fusion of PP with  emm6 , was introduced by natural transformation in  S. gordonii  GP201, a strain with a single copy of Tn 916  integrated into the chromosome. One of the transformants (GP1241), in which the integrative suicide vector drove the integration of the PP- emm6  fusion into Tn 916 , was isolated and analyzed for M6 protein expression. Envelope fractions (containing surface associated proteins) prepared from equal amounts of cells grown to mid-log, early and late stationary phase, were analyzed by Western-blotting with an anti-M6 monoclonal antibody. Multiple bands could be detected in fractions of cells grown to mid-log and early stationary phase, whereas no band was detected in the fraction of cultures grown to late stationary phase (Fig.  6A ). The intensity of the signal was higher during the mid-log growth phase suggesting that either PP is more active during exponential growth or that the M6 protein is being degraded during stationary phase. The presence of multiple reactive bands of molecular masses close to the hypothetical size of M6 (predicted molecular weight, 49 kDa) is probably due to the fact that coiled-coil proteins like M6 run at aberrant sizes on denaturing gels [ 19 ]. Expression of M6/TTFC in  B. subtilis Competent cells of  B. subtilis  GP800.2, containing one copy of Tn 916  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 Using a previously described genetic system [ 12 ], we constructed a derivative of the  E. faecalis  strain OG1SS [ 20 ] expressing the recombinant M6/OVA protein under PP control. pSMB156, bearing the PP- emm6/ova  fusion, was first introduced in the Tn 916  containing  B. subtilis  GP800.2 by natural transformation, to obtain a recombinant conjugative transposon containing the transcriptional fusion. The recombinant transposon was then transferred by conjugation into  E. faecalis  OG1SS. Transconjugants were detected at a frequency of 4 × 10 -10  transconjugants/recipient. One of them (GP431) was analyzed for cell-surface expression of M6/OVA by flow-cytometric analysis using an anti-ovalbumin polyclonal antibody. The presence of recombinant M6/OVA on the surface of GP431 was clearly demonstrated by the increase of the fluorescence intensity in this strain, as compared to the parental control OG1SS (Fig.  7 ).
Results and discussion
Promoter selection by chromosomal integration To select resident promoters from the genome of  Streptococcus gordonii , we performed a random ligation of streptococcal DNA to a promoterless  cat  gene, conferring resistance to chloramphenicol (Cm). The ligation mixture was used to transform the naturally transformable  S. gordonii  «Challis» strain V288 and transformants were selected for Cm resistance. Chromosomal DNA flanking the promoterless  cat  gene provided the homology for the random integration of  cat  into the chromosome during transformation (Fig.  1 ). 71 Cm-resistant (Cm R ) transformants were isolated, presumably as a result of transcriptional fusions of streptococcal promoters to the promoterless  cat  gene. Eighteen Cm R  transformants were selected for further characterization. The strategy commonly used to select promoters is based on cloning random chromosomal DNA fragments in a promoter probe vector upstream of a promoterless reporter gene. However, integrating the promoterless reporter gene ( cat ) directly into the streptococcal chromosome, allowed us to select resident chromosomal promoters expressing  cat  after  in vivo  transcriptional fusion at a single locus on the chromosome. This is preferable when looking for promoters to express heterologous genes integrated into the chromosome in a single copy. 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.
Promoter selection by chromosomal integration
To select resident promoters from the genome of  Streptococcus gordonii , we performed a random ligation of streptococcal DNA to a promoterless  cat  gene, conferring resistance to chloramphenicol (Cm). The ligation mixture was used to transform the naturally transformable  S. gordonii  «Challis» strain V288 and transformants were selected for Cm resistance. Chromosomal DNA flanking the promoterless  cat  gene provided the homology for the random integration of  cat  into the chromosome during transformation (Fig.  1 ). 71 Cm-resistant (Cm R ) transformants were isolated, presumably as a result of transcriptional fusions of streptococcal promoters to the promoterless  cat  gene. Eighteen Cm R  transformants were selected for further characterization. The strategy commonly used to select promoters is based on cloning random chromosomal DNA fragments in a promoter probe vector upstream of a promoterless reporter gene. However, integrating the promoterless reporter gene ( cat ) directly into the streptococcal chromosome, allowed us to select resident chromosomal promoters expressing  cat  after  in vivo  transcriptional fusion at a single locus on the chromosome. This is preferable when looking for promoters to express heterologous genes integrated into the chromosome in a single copy.Streptococcus gordoniicatS. gordoniicatcatcatcatcatin vivoIn 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.In vivoCloning 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  Taq I 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 Cm R . 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  Taq I 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 Cm R  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 [ 15 ].
Cloning of a promoter from  S. gordonii
S. gordoniiThe 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  Taq I 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 Cm R . 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  Taq I 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 Cm R  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 [ 15 ].catcatcatTaqcatEscherichia coliTaqcatE. coliE. coliSequence analysis The streptococcal DNA upstream of the  cat  gene in pVMB5 was sequenced (GenBank accession number: U74080). Analysis of the sequence revealed the presence of an open reading frame (ORF1), preceded by a typical ribosome binding site (RBS) (Fig.  2 ). A BLAST search with the partial sequenced genome of  S. gordonii   showed that ORF1 represents the 5'-end of a gene encoding the first 48 amino acids (aa) of an uncharacterized protein conserved in bacteria and whose function is unknown. As indicated in figure  2 , this truncated streptococcal protein is translationally fused to the N-terminus of CAT. Sequence analysis also revealed that the first 122 bp of the cloned streptococcal fragment belong to the 3'-end of a gene encoding a putative acetyltransferase (ORF2). To obtain information about the chromosomal region containing the 600 bp streptococcal DNA fragment cloned in pVMB5, we looked throughout the whole contig sequence and we found out that upstream ORF2, and partially overlapping with it, there is an ORF encoding a DltD horthologue ( dltD ), a protein involved in D-alanine incorporation into lipoteichoic acid (LTA) [ 16 ], whereas downstream ORF1, there is a gene encoding a Tmp7 transmembrane protein (Fig.  2 ).
Sequence analysis
The streptococcal DNA upstream of the  cat  gene in pVMB5 was sequenced (GenBank accession number: U74080). Analysis of the sequence revealed the presence of an open reading frame (ORF1), preceded by a typical ribosome binding site (RBS) (Fig.  2 ). A BLAST search with the partial sequenced genome of  S. gordonii   showed that ORF1 represents the 5'-end of a gene encoding the first 48 amino acids (aa) of an uncharacterized protein conserved in bacteria and whose function is unknown. As indicated in figure  2 , this truncated streptococcal protein is translationally fused to the N-terminus of CAT. Sequence analysis also revealed that the first 122 bp of the cloned streptococcal fragment belong to the 3'-end of a gene encoding a putative acetyltransferase (ORF2). To obtain information about the chromosomal region containing the 600 bp streptococcal DNA fragment cloned in pVMB5, we looked throughout the whole contig sequence and we found out that upstream ORF2, and partially overlapping with it, there is an ORF encoding a DltD horthologue ( dltD ), a protein involved in D-alanine incorporation into lipoteichoic acid (LTA) [ 16 ], whereas downstream ORF1, there is a gene encoding a Tmp7 transmembrane protein (Fig.  2 ).catS. gordoniidltDIdentification of the transcriptional start site To identify the sequences responsible for the observed promoter activity, the 5'-end of the  cat- specific mRNA was mapped by primer extension with a specific primer. Total RNA was isolated from  E. coli  GP334 (containing pVMB5) and  S. gordonii  GP215. In both strains, the position of the 5'-end of the mRNA was located at the same purine residue at position 423 of the 600 bp region of streptococcal DNA, 26 nucleotides upstream of the ORF1 translational start site (Fig.  2  and  3 ). Putative -35 and -10 sequences closely resembling the consensus  E. coli  σ 70  and  Bacillus subtilis  σ 43  [ 17 ] recognition sequences TTGACA (-35) and TATAAT (-10) could be identified. The -35 region was TTGCAA, and the -10 region was TAGAAT. The spacing between the -35 and -10 region was 17 bp and was thus similar to the spacing in  B. subtilis  (17 to 19 bp) and  E. coli  (16 to 18 bp) promoters. Moreover, a TG nucleotide pair was found 1 bp upstream of the -10 region; such a structure is typical of Gram-positive bacteria promoters [ 18 ]. Based on this information, we concluded that indeed we isolated a streptococcal promoter which was designated PP. The spacing between the 5'-end of the mRNA and the -10 hexanucleotide was only 2 bp, which is unusually short. A second putative -10 region could be identified in the TATGAT hexanucleotide (Fig.  2 ), whose distance from the 5' end of the mRNA is 7 bp. However, since this sequence is not preceded by the TG nucleotide pair typical of Gram-positive promoters, and is separated from the -35 region only by 12 bp, we suppose that this -10 region of PP is probably not active.
Identification of the transcriptional start site
To identify the sequences responsible for the observed promoter activity, the 5'-end of the  cat- specific mRNA was mapped by primer extension with a specific primer. Total RNA was isolated from  E. coli  GP334 (containing pVMB5) and  S. gordonii  GP215. In both strains, the position of the 5'-end of the mRNA was located at the same purine residue at position 423 of the 600 bp region of streptococcal DNA, 26 nucleotides upstream of the ORF1 translational start site (Fig.  2  and  3 ). Putative -35 and -10 sequences closely resembling the consensus  E. coli  σ 70  and  Bacillus subtilis  σ 43  [ 17 ] recognition sequences TTGACA (-35) and TATAAT (-10) could be identified. The -35 region was TTGCAA, and the -10 region was TAGAAT. The spacing between the -35 and -10 region was 17 bp and was thus similar to the spacing in  B. subtilis  (17 to 19 bp) and  E. coli  (16 to 18 bp) promoters. Moreover, a TG nucleotide pair was found 1 bp upstream of the -10 region; such a structure is typical of Gram-positive bacteria promoters [ 18 ]. Based on this information, we concluded that indeed we isolated a streptococcal promoter which was designated PP. The spacing between the 5'-end of the mRNA and the -10 hexanucleotide was only 2 bp, which is unusually short. A second putative -10 region could be identified in the TATGAT hexanucleotide (Fig.  2 ), whose distance from the 5' end of the mRNA is 7 bp. However, since this sequence is not preceded by the TG nucleotide pair typical of Gram-positive promoters, and is separated from the -35 region only by 12 bp, we suppose that this -10 region of PP is probably not active.cat-E. coliS. gordoniiE. coliBacillus subtilisB. subtilisE. coliInsertion vectors to express heterologous proteins We have previously developed a genetic system based on the use of  Streptococcus pyogenes  surface fibrillar M6 protein, as a partner for the construction of translational fusions to deliver foreign proteins on the surface of  S. gordonii  [ 10 ]. To determine the possibility of using PP for chromosomal single copy heterologous gene expression in Gram-positive bacteria, we first generated a transcriptional fusion of PP with a promoterless gene encoding the M6 protein ( emm6 ) in pSMB47, a suicide vector capable of integrating heterologous DNA into the conjugative transposon Tn 916  via homologous recombinantion [ 12 ]. The recombinant plasmid was named pSMB139 (see Methods) (Fig.  4 ). To construct a Tn 916  insertion vector that could be used to express translational fusions between M6 and any heterologous proteins under the PP promoter control, a 900 bp  Avr II- Hind III fragment internal to  emm6  in pSMB139 was replaced by a 390 bp  Avr II- Hind III fragment of pSMB55 containing a multiple cloning site [ 10 ] (Fig.  4 ). The resulting vector, named pSMB148 (Fig.  4 ), allows to create translational fusions of heterologous proteins between the first 122 N-terminal aa and the last 140 aa of the M6 protein, which provides sequences necessary for cell wall anchoring. pSMB148 was used to create two derivative vectors in which the  emm6  gene was fused respectively with a DNA sequence encoding 339 aa of the chicken ovalbumin (OVA) (pSMB156), and a DNA sequence encoding 458 aa of the tetanus toxin fragment C (TTFC) (pSMB288) (see Methods). A schematic representation of the recombinant proteins is shown in Fig.  5A .
Insertion vectors to express heterologous proteins
We have previously developed a genetic system based on the use of  Streptococcus pyogenes  surface fibrillar M6 protein, as a partner for the construction of translational fusions to deliver foreign proteins on the surface of  S. gordonii  [ 10 ]. To determine the possibility of using PP for chromosomal single copy heterologous gene expression in Gram-positive bacteria, we first generated a transcriptional fusion of PP with a promoterless gene encoding the M6 protein ( emm6 ) in pSMB47, a suicide vector capable of integrating heterologous DNA into the conjugative transposon Tn 916  via homologous recombinantion [ 12 ]. The recombinant plasmid was named pSMB139 (see Methods) (Fig.  4 ). To construct a Tn 916  insertion vector that could be used to express translational fusions between M6 and any heterologous proteins under the PP promoter control, a 900 bp  Avr II- Hind III fragment internal to  emm6  in pSMB139 was replaced by a 390 bp  Avr II- Hind III fragment of pSMB55 containing a multiple cloning site [ 10 ] (Fig.  4 ). The resulting vector, named pSMB148 (Fig.  4 ), allows to create translational fusions of heterologous proteins between the first 122 N-terminal aa and the last 140 aa of the M6 protein, which provides sequences necessary for cell wall anchoring.Streptococcus pyogenesS. gordoniiemm6916916AvrHindemm6AvrHindpSMB148 was used to create two derivative vectors in which the  emm6  gene was fused respectively with a DNA sequence encoding 339 aa of the chicken ovalbumin (OVA) (pSMB156), and a DNA sequence encoding 458 aa of the tetanus toxin fragment C (TTFC) (pSMB288) (see Methods). A schematic representation of the recombinant proteins is shown in Fig.  5A .emm6Expression 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 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.S. gordoniiB. subtilisEnterococcus faecalisExpression of M6 in  S. gordonii pSMB139, bearing a transcriptional fusion of PP with  emm6 , was introduced by natural transformation in  S. gordonii  GP201, a strain with a single copy of Tn 916  integrated into the chromosome. One of the transformants (GP1241), in which the integrative suicide vector drove the integration of the PP- emm6  fusion into Tn 916 , was isolated and analyzed for M6 protein expression. Envelope fractions (containing surface associated proteins) prepared from equal amounts of cells grown to mid-log, early and late stationary phase, were analyzed by Western-blotting with an anti-M6 monoclonal antibody. Multiple bands could be detected in fractions of cells grown to mid-log and early stationary phase, whereas no band was detected in the fraction of cultures grown to late stationary phase (Fig.  6A ). The intensity of the signal was higher during the mid-log growth phase suggesting that either PP is more active during exponential growth or that the M6 protein is being degraded during stationary phase. The presence of multiple reactive bands of molecular masses close to the hypothetical size of M6 (predicted molecular weight, 49 kDa) is probably due to the fact that coiled-coil proteins like M6 run at aberrant sizes on denaturing gels [ 19 ].
Expression of M6 in  S. gordonii
S. gordoniipSMB139, bearing a transcriptional fusion of PP with  emm6 , was introduced by natural transformation in  S. gordonii  GP201, a strain with a single copy of Tn 916  integrated into the chromosome. One of the transformants (GP1241), in which the integrative suicide vector drove the integration of the PP- emm6  fusion into Tn 916 , was isolated and analyzed for M6 protein expression. Envelope fractions (containing surface associated proteins) prepared from equal amounts of cells grown to mid-log, early and late stationary phase, were analyzed by Western-blotting with an anti-M6 monoclonal antibody. Multiple bands could be detected in fractions of cells grown to mid-log and early stationary phase, whereas no band was detected in the fraction of cultures grown to late stationary phase (Fig.  6A ). The intensity of the signal was higher during the mid-log growth phase suggesting that either PP is more active during exponential growth or that the M6 protein is being degraded during stationary phase. The presence of multiple reactive bands of molecular masses close to the hypothetical size of M6 (predicted molecular weight, 49 kDa) is probably due to the fact that coiled-coil proteins like M6 run at aberrant sizes on denaturing gels [ 19 ].emm6S. gordonii916emm6916Expression of M6/TTFC in  B. subtilis Competent cells of  B. subtilis  GP800.2, containing one copy of Tn 916  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/TTFC in  B. subtilis
B. subtilisCompetent cells of  B. subtilis  GP800.2, containing one copy of Tn 916  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).B. subtilis916emm6/ttfcExpression of M6/OVA in  E. faecalis Using a previously described genetic system [ 12 ], we constructed a derivative of the  E. faecalis  strain OG1SS [ 20 ] expressing the recombinant M6/OVA protein under PP control. pSMB156, bearing the PP- emm6/ova  fusion, was first introduced in the Tn 916  containing  B. subtilis  GP800.2 by natural transformation, to obtain a recombinant conjugative transposon containing the transcriptional fusion. The recombinant transposon was then transferred by conjugation into  E. faecalis  OG1SS. Transconjugants were detected at a frequency of 4 × 10 -10  transconjugants/recipient. One of them (GP431) was analyzed for cell-surface expression of M6/OVA by flow-cytometric analysis using an anti-ovalbumin polyclonal antibody. The presence of recombinant M6/OVA on the surface of GP431 was clearly demonstrated by the increase of the fluorescence intensity in this strain, as compared to the parental control OG1SS (Fig.  7 ).
Expression of M6/OVA in  E. faecalis
E. faecalisUsing a previously described genetic system [ 12 ], we constructed a derivative of the  E. faecalis  strain OG1SS [ 20 ] expressing the recombinant M6/OVA protein under PP control. pSMB156, bearing the PP- emm6/ova  fusion, was first introduced in the Tn 916  containing  B. subtilis  GP800.2 by natural transformation, to obtain a recombinant conjugative transposon containing the transcriptional fusion. The recombinant transposon was then transferred by conjugation into  E. faecalis  OG1SS. Transconjugants were detected at a frequency of 4 × 10 -10  transconjugants/recipient. One of them (GP431) was analyzed for cell-surface expression of M6/OVA by flow-cytometric analysis using an anti-ovalbumin polyclonal antibody. The presence of recombinant M6/OVA on the surface of GP431 was clearly demonstrated by the increase of the fluorescence intensity in this strain, as compared to the parental control OG1SS (Fig.  7 ).E. faecalisemm6/ova916B. subtilisE. faecalisConclusions 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.
Conclusions
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.S. gordoniiMethods Bacterial strains, plasmids and growth conditions All strains and plasmids used in this work are listed in Table  1 .  E. coli  strains DH5α and HB101 were cultured in Luria-Bertani (LB) broth. For maintenance of plasmids, ampicillin (100 μg/ml), chloramphenicol (20 μg/ml) or erythromycin (100 μg/ml) was added to the growth medium. Streptococcal strains were cultured in Brain Heart Infusion medium (BHI, Difco) or Tryptic Soy Broth (TSB, Difco) in the presence of chloramphenicol (5 μg/ml), erythromicin (100 μg/ml) or streptomycin (500 μg/ml) whenever required. Transformation of naturally competent cells of  S. gordonii  V288 and GP201, scoring and genetic analysis of transformants was carried out as already described [ 21 , 22 ].  B. subtilis  strains were grown in LB broth with erythromicin (3 μg/ml) when it was required. Competent cells of  B. subtilis  GP800.2 were prepared and transformed according to described procedures [ 23 ]. Agar (1.5%) was added to LB, BHI or TSB to obtain solid media. All cultures were incubated at 37°C. DNA manipulation Total DNA preparation of  S. gordonii  was performed as previously described [ 21 ]. Plasmid DNA was prepared using the Qiagen Plasmid Kit (Qiagen) according to the manufacturer's instructions. All recombinant techniques were performed following standard procedures [ 24 ], 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  Hind III- Bam HI fragment of plasmid pKT [ 25 ], containing a  cat  gene, was ligated with random chromosomal DNA fragments of  S. gordonii  V288 previously cut with both  Hind III and  Bam HI. The initiation codon of  cat  was 34 bp downstream of the  Hind III site, therefore  Hind III 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 Cm R . MIC determination The MIC of chloramphenicol for  S. gordonii  was determined following standard procedures [ 26 ]. 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 [ 22 ] using the oligonucleotides 5'-AT GGATCC AT CATATG GCTAAAAATAACACGAAT-3' (upstream primer, containing a  Bam HI site and introducing a  Nde I site at the ATG translation initiation codon) and 5'-GCAT GTCGAC CATAATCATTAAATGTATCTCAT-3' (downstream primers containing a  Sal I site). This 1648 bp PCR fragment was digested with  Bam HI and  Sal I and cloned in pVA891 [ 27 ] previously digested with  Bam HIand  Sal I, resulting in plasmid pSMB89. A 451 bp region containing the streptococcal promoter PP was amplified from pVMB5 using the following primers: 5'-CGA GGATCC TTTA ATCGAT ACTCATG-3' (upstream primer, containing a  Bam HI and a  Cla I site) and 5'-CCG CATATG GTTCTCCTTTTTATTTGT-3' (downstream primers containing a  Nde I site). After digestion with  Bam HI and  Nde I, this PCR product was inserted between the  Bam HI and  Nde I site of pSMB89 to obtain a transcriptional fusion of PP with  emm6 . The resulting plasmid was named pSMB128. This plasmid was first cut with  Bam HI, treated with Klenow enzyme to generate blunt ends, and finally cut with  Sal I to obtain a 2.0 kbfragment containing PP- emm6  fusion. This fragment was gel-purified and ligated to the suicide integrative plasmid pSMB47 [ 12 ] previously cut with  Hind III, treated with Klenow enzyme to generate blunt ends, and finally cut with  Sal I. The resulting plasmid was named pSMB139 (Fig.  4 ). To introduce a multiple cloning site in the  emm6  gene contained in pSMB139, the 900 bp  Avr II- Hind III fragment internal to  emm6  was replaced with the 390 bp  Avr II- Hind III fragment of  emm6  from pSMB55 [ 10 ] (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'-CT AGATCT GACAGCA CCAGGACAC-3' (upstream primer containing a  Bgl II site) and 5'-TA AAGCTT TAGGGG AAACACATCTG-3' (downstream primer containing a  Hind III site). After digestion with  Bgl II and  Hind III, this segment was introduced in pSMB148 previously digested with  Bgl II and  Hind III. 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  Bgl II- Hind III fragment encoding 458 aa of TTFC was isolated from pSMB158 [ 28 ] and cloned between the  Bgl II and  Hind III sites of pSMB148. The resulting plasmid was named pSMB288. Western-blot analysis 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 [ 31 ] 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. Flow-cytometric analysis Flow-cytometric analysis of  E. faecalis  was performed as already described [ 28 , 32 ] using an anti-OVA rabbit serum diluted 1:300 [ 29 ]. DNA sequence determination The promoter containing fragment cloned in pVMB5 was sequenced by dideoxy chain termination method [ 33 ] as already described [ 34 ]. Denatured plasmid DNA was used as template. RNA isolation Total RNA was isolated from a 50 ml cell culture of  S. gordonii  and  E. coli  grown to late exponential phase (OD 590  ≌ 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 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- 32 P] ATP (3000 Ci/mmol, Amersham) using T 4  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 MgCl 2 , 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 Conjugation experiments were performed on solid media as previously described [ 12 ].
Methods
Bacterial strains, plasmids and growth conditions All strains and plasmids used in this work are listed in Table  1 .  E. coli  strains DH5α and HB101 were cultured in Luria-Bertani (LB) broth. For maintenance of plasmids, ampicillin (100 μg/ml), chloramphenicol (20 μg/ml) or erythromycin (100 μg/ml) was added to the growth medium. Streptococcal strains were cultured in Brain Heart Infusion medium (BHI, Difco) or Tryptic Soy Broth (TSB, Difco) in the presence of chloramphenicol (5 μg/ml), erythromicin (100 μg/ml) or streptomycin (500 μg/ml) whenever required. Transformation of naturally competent cells of  S. gordonii  V288 and GP201, scoring and genetic analysis of transformants was carried out as already described [ 21 , 22 ].  B. subtilis  strains were grown in LB broth with erythromicin (3 μg/ml) when it was required. Competent cells of  B. subtilis  GP800.2 were prepared and transformed according to described procedures [ 23 ]. Agar (1.5%) was added to LB, BHI or TSB to obtain solid media. All cultures were incubated at 37°C.
Bacterial strains, plasmids and growth conditions
All strains and plasmids used in this work are listed in Table  1 .  E. coli  strains DH5α and HB101 were cultured in Luria-Bertani (LB) broth. For maintenance of plasmids, ampicillin (100 μg/ml), chloramphenicol (20 μg/ml) or erythromycin (100 μg/ml) was added to the growth medium. Streptococcal strains were cultured in Brain Heart Infusion medium (BHI, Difco) or Tryptic Soy Broth (TSB, Difco) in the presence of chloramphenicol (5 μg/ml), erythromicin (100 μg/ml) or streptomycin (500 μg/ml) whenever required. Transformation of naturally competent cells of  S. gordonii  V288 and GP201, scoring and genetic analysis of transformants was carried out as already described [ 21 , 22 ].  B. subtilis  strains were grown in LB broth with erythromicin (3 μg/ml) when it was required. Competent cells of  B. subtilis  GP800.2 were prepared and transformed according to described procedures [ 23 ]. Agar (1.5%) was added to LB, BHI or TSB to obtain solid media. All cultures were incubated at 37°C.E. coliS. gordoniiB. subtilisB. subtilisDNA manipulation Total DNA preparation of  S. gordonii  was performed as previously described [ 21 ]. Plasmid DNA was prepared using the Qiagen Plasmid Kit (Qiagen) according to the manufacturer's instructions. All recombinant techniques were performed following standard procedures [ 24 ], using  E. coli  HB101 or DH5α as a host. DNA restriction enzymes were obtained from Roche and used according to the manufacturer's instructions.
DNA manipulation
Total DNA preparation of  S. gordonii  was performed as previously described [ 21 ]. Plasmid DNA was prepared using the Qiagen Plasmid Kit (Qiagen) according to the manufacturer's instructions. All recombinant techniques were performed following standard procedures [ 24 ], using  E. coli  HB101 or DH5α as a host. DNA restriction enzymes were obtained from Roche and used according to the manufacturer's instructions.S. gordoniiE. coliPromoter selection by chromosomal integration A 1.6 kb  Hind III- Bam HI fragment of plasmid pKT [ 25 ], containing a  cat  gene, was ligated with random chromosomal DNA fragments of  S. gordonii  V288 previously cut with both  Hind III and  Bam HI. The initiation codon of  cat  was 34 bp downstream of the  Hind III site, therefore  Hind III 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 Cm R .
Promoter selection by chromosomal integration
A 1.6 kb  Hind III- Bam HI fragment of plasmid pKT [ 25 ], containing a  cat  gene, was ligated with random chromosomal DNA fragments of  S. gordonii  V288 previously cut with both  Hind III and  Bam HI. The initiation codon of  cat  was 34 bp downstream of the  Hind III site, therefore  Hind III 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 Cm R .HindBamcatS. gordoniiHindBamcatHindHindcatS. gordoniiMIC determination The MIC of chloramphenicol for  S. gordonii  was determined following standard procedures [ 26 ].
MIC determination
The MIC of chloramphenicol for  S. gordonii  was determined following standard procedures [ 26 ].S. gordoniiConstruction 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 [ 22 ] using the oligonucleotides 5'-AT GGATCC AT CATATG GCTAAAAATAACACGAAT-3' (upstream primer, containing a  Bam HI site and introducing a  Nde I site at the ATG translation initiation codon) and 5'-GCAT GTCGAC CATAATCATTAAATGTATCTCAT-3' (downstream primers containing a  Sal I site). This 1648 bp PCR fragment was digested with  Bam HI and  Sal I and cloned in pVA891 [ 27 ] previously digested with  Bam HIand  Sal I, resulting in plasmid pSMB89. A 451 bp region containing the streptococcal promoter PP was amplified from pVMB5 using the following primers: 5'-CGA GGATCC TTTA ATCGAT ACTCATG-3' (upstream primer, containing a  Bam HI and a  Cla I site) and 5'-CCG CATATG GTTCTCCTTTTTATTTGT-3' (downstream primers containing a  Nde I site). After digestion with  Bam HI and  Nde I, this PCR product was inserted between the  Bam HI and  Nde I site of pSMB89 to obtain a transcriptional fusion of PP with  emm6 . The resulting plasmid was named pSMB128. This plasmid was first cut with  Bam HI, treated with Klenow enzyme to generate blunt ends, and finally cut with  Sal I to obtain a 2.0 kbfragment containing PP- emm6  fusion. This fragment was gel-purified and ligated to the suicide integrative plasmid pSMB47 [ 12 ] previously cut with  Hind III, treated with Klenow enzyme to generate blunt ends, and finally cut with  Sal I. The resulting plasmid was named pSMB139 (Fig.  4 ). To introduce a multiple cloning site in the  emm6  gene contained in pSMB139, the 900 bp  Avr II- Hind III fragment internal to  emm6  was replaced with the 390 bp  Avr II- Hind III fragment of  emm6  from pSMB55 [ 10 ] (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'-CT AGATCT GACAGCA CCAGGACAC-3' (upstream primer containing a  Bgl II site) and 5'-TA AAGCTT TAGGGG AAACACATCTG-3' (downstream primer containing a  Hind III site). After digestion with  Bgl II and  Hind III, this segment was introduced in pSMB148 previously digested with  Bgl II and  Hind III. 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  Bgl II- Hind III fragment encoding 458 aa of TTFC was isolated from pSMB158 [ 28 ] and cloned between the  Bgl II and  Hind III sites of pSMB148. The resulting plasmid was named pSMB288.
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 [ 22 ] using the oligonucleotides 5'-AT GGATCC AT CATATG GCTAAAAATAACACGAAT-3' (upstream primer, containing a  Bam HI site and introducing a  Nde I site at the ATG translation initiation codon) and 5'-GCAT GTCGAC CATAATCATTAAATGTATCTCAT-3' (downstream primers containing a  Sal I site). This 1648 bp PCR fragment was digested with  Bam HI and  Sal I and cloned in pVA891 [ 27 ] previously digested with  Bam HIand  Sal I, resulting in plasmid pSMB89. A 451 bp region containing the streptococcal promoter PP was amplified from pVMB5 using the following primers: 5'-CGA GGATCC TTTA ATCGAT ACTCATG-3' (upstream primer, containing a  Bam HI and a  Cla I site) and 5'-CCG CATATG GTTCTCCTTTTTATTTGT-3' (downstream primers containing a  Nde I site). After digestion with  Bam HI and  Nde I, this PCR product was inserted between the  Bam HI and  Nde I site of pSMB89 to obtain a transcriptional fusion of PP with  emm6 . The resulting plasmid was named pSMB128. This plasmid was first cut with  Bam HI, treated with Klenow enzyme to generate blunt ends, and finally cut with  Sal I to obtain a 2.0 kbfragment containing PP- emm6  fusion. This fragment was gel-purified and ligated to the suicide integrative plasmid pSMB47 [ 12 ] previously cut with  Hind III, treated with Klenow enzyme to generate blunt ends, and finally cut with  Sal I. The resulting plasmid was named pSMB139 (Fig.  4 ). To introduce a multiple cloning site in the  emm6  gene contained in pSMB139, the 900 bp  Avr II- Hind III fragment internal to  emm6  was replaced with the 390 bp  Avr II- Hind III fragment of  emm6  from pSMB55 [ 10 ] (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'-CT AGATCT GACAGCA CCAGGACAC-3' (upstream primer containing a  Bgl II site) and 5'-TA AAGCTT TAGGGG AAACACATCTG-3' (downstream primer containing a  Hind III site). After digestion with  Bgl II and  Hind III, this segment was introduced in pSMB148 previously digested with  Bgl II and  Hind III. 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  Bgl II- Hind III fragment encoding 458 aa of TTFC was isolated from pSMB158 [ 28 ] and cloned between the  Bgl II and  Hind III sites of pSMB148. The resulting plasmid was named pSMB288.emm6S. pyogenesBamNdeSalBamSalBamSalBamClaNdeBamNdeBamNdeemm6BamSalemm6HindSalemm6AvrHindemm6AvrHindemm6BglHindBglHindBglHindBglHindBglHindWestern-blot analysis 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 [ 31 ] 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.
Western-blot analysis
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 [ 31 ] 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.S. gordoniiB. subtilisFlow-cytometric analysis Flow-cytometric analysis of  E. faecalis  was performed as already described [ 28 , 32 ] using an anti-OVA rabbit serum diluted 1:300 [ 29 ].
Flow-cytometric analysis
Flow-cytometric analysis of  E. faecalis  was performed as already described [ 28 , 32 ] using an anti-OVA rabbit serum diluted 1:300 [ 29 ].E. faecalisDNA sequence determination The promoter containing fragment cloned in pVMB5 was sequenced by dideoxy chain termination method [ 33 ] as already described [ 34 ]. Denatured plasmid DNA was used as template.
DNA sequence determination
The promoter containing fragment cloned in pVMB5 was sequenced by dideoxy chain termination method [ 33 ] as already described [ 34 ]. Denatured plasmid DNA was used as template.RNA isolation Total RNA was isolated from a 50 ml cell culture of  S. gordonii  and  E. coli  grown to late exponential phase (OD 590  ≌ 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).
RNA isolation
Total RNA was isolated from a 50 ml cell culture of  S. gordonii  and  E. coli  grown to late exponential phase (OD 590  ≌ 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).S. gordoniiE. coligE. coliS. gordoniiPrimer-extention analysis 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- 32 P] ATP (3000 Ci/mmol, Amersham) using T 4  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 MgCl 2 , 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.
Primer-extention analysis
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- 32 P] ATP (3000 Ci/mmol, Amersham) using T 4  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 MgCl 2 , 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.catConjugation Conjugation experiments were performed on solid media as previously described [ 12 ].
Conjugation
Conjugation experiments were performed on solid media as previously described [ 12 ].Authors' contributions RP, characterization of promoter, engineering of  S. gordonii , writing of manuscript. TM, engineering of  B. subtilis  and  E. faecalis MRO, participation in experimental work, data evaluation RM, participation in experimental work, data evaluation, writing of manuscript GP, design and coordination of the study, data evaluation, direct supervision of experimental work, writing of manuscript All authors read and approved the final manuscript.
Authors' contributions
RP, characterization of promoter, engineering of  S. gordonii , writing of manuscript.S. gordoniiTM, engineering of  B. subtilis  and  E. faecalisB. subtilisE. faecalisMRO, participation in experimental work, data evaluationRM, participation in experimental work, data evaluation, writing of manuscriptGP, design and coordination of the study, data evaluation, direct supervision of experimental work, writing of manuscriptAll authors read and approved the final manuscript.