Selection and characterization of a promoter for expression of single-copy recombinant genes in Gram-positive bacteria

Abstract

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 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.

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.

Figure 1

In vivo transcriptional fusion by integration of a promoterless cat gene into the chromosome of a naturally transformable streptococcus. A promoterless cat gene is ligated in vitro to random chromosomal fragments, using a restriction site a few base pairs upstream of its translation initiation codon (a). The ligation mixture is then used to transform the recipient strain (b), and the homologous sequences allow chromosomal integration of the promoterless cat gene (c). By this process, the cat gene is integrated into the chromosome between two direct repeats. Since some of the chromosomal fragments contain promoters (P), it is possible to obtain expression of the promoterless cat gene after in vivo transcriptional fusion with resident chromosomal promoters.

Figure 2

Schematic representation of the S. gordonii locus containing PP promoter. DltD orthologue, an ORF encoding a putative acetyltransferase (ORF2), ORF1, the gene encoding a Tmp7 transmembrane protein, and the cat gene are indicated by arrows. Dashed arrows designate that the ORF is only partially represented in the scheme. The 600 bp TaqI-HindIII fragment including PP promoter and cloned upstream of the cat gene in pVMB5 is indicated by a dotted box. Nucleotide sequence of the PP promoter region (332 bp) is reported inside the box. The transcriptional start site determined by primer extention analysis is marked with an asterisk. Proposed -35 and -10 regions are underlined. A second putative -10 sequence is overlined. ORF1 putative ribosome binding site (RBS) is boxed. ATG initiation codon of cat is in bold characters and the sequence of HindIII site is in italic letters. The complete sequence of the 600 bp cloned fragment is available on GenBank (Accession number: U74080). T, TaqI site; B, BamHI site; H, HindIII site ; loop, putative transcriptional terminator.

Figure 3

Primer extension analysis of the PP promoter. Localization by primer extension of the transcriptional start site of the cat mRNA specified by the PP promoter in E. coli GP334 (lanes 1–2), and S. gordonii GP215 (lanes 3–4). The sequence of the region upstream of the cat gene in pVMB5 was used as standard. The A residue, complementary to the T at position 423, indicated by an arrow, represents the transcriptional start site used in both strains.

Figure 4

Construction of the insertion plasmid pSMB148. pSMB139 was constructed by introducing a 2.0 kb fragment, containing a transcriptional fusion of PP promoter with emm6, in the Tn916 insertion vector pSMB47 (see Methods). pSMB148 is a derivative of pSMB139 in which a 900 bp AvrII-HindIII fragment was replaced with a 390 bp AvrII-HindIII fragment from pSMB55 containing a multiple cloning site [10].

Figure 5

Schematic representation of recombinant M6, M6/TTFC and M6/OVA expressed on the surface of S. gordonii, B. subtilis and E. faecalis. The 458 aa protein TTFC (white bar) and the 339 aa protein OVA (light gray bar) were fused with the first 122 N-terminal aminoacids and the last 140 C-terminal aminoacids of M6 (dark gray bar). The predicted molecular weight of M6, M6/TTFC and M6/OVA is 49 kDa, 82 kDa and 66.9 kDa respectively.

Figure 6

Western blot analysis of recombinant S. gordonii and B. subtilis strains expressing M6 protein and M6/TTFC fusion protein. (A) S. gordonii envelope fractions. Lane 1 through 3, GP1241 expressing M6 under the control of PP promoter. Lane 1, GP1241 harvested after overnight growth. Lane 2, GP1241 harvested after early stationary phase. Lane 3, GP1241 harvested after exponential phase. Lane 4, recipient strain GP201 (negative control). Lane 5, GP231 (positive control). Blot was developed with anti-M6 monoclonal antibody 10B6. (B) S. gordonii and B. subtilis envelope fractions. Lane 1, S. gordonii GP204 (negative control). Lane 2, S. gordonii GP1253 expressing M6/TTFC (positive control). Lane 3, B. subtilis recipient strain GP800.2 (negative control). Lane 4, B. subtilis GP848 expressing M6/TTFC under the control of PP promoter. Blot was developed with anti TTFC rabbit serum. Molecular weight markers are shown in the right side of panels.

Figure 7

Flow-cytometric analysis of E. faecalis expressing M6/OVA. (A) OG1SS, recipient strain not expressing M6/OVA. (B) GP431, recombinant strain expressing M6/OVA on the surface. Bacterial cells were treated with anti-OVA rabbit serum and than with FITC-conjugated goat anti-rabbit IgG. x axis, arbitrary units (a.u.) of fluorescence intensity (log₁₀); y axis, relative cell number.