Selection and characterization of a promoter for expression of single-copy recombinant genes in 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 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.


CONCLUSIONS
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.
Provvedi
Roberta
R
Laboratory of Molecular Microbiology and Biotechnology, Department of Molecular Biology, University of Siena, Policlinico Le Scotte, Viale Bracci, 53100 Siena, Italy. roberta.provvedi@unipd.it
Maggi
Tiziana
T
Oggioni
Marco R
MR
Manganelli
Riccardo
R
Pozzi
Gianni
G
D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001412:Bacillus subtilis / Q000235:genetics; D001431:Bacteriological Techniques; D001709:Biotechnology / Q000379:methods*; D015153:Blotting, Western; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics; D002876:Chromosomes, Bacterial; D003001:Cloning, Molecular; D017931:DNA Primers / Q000737:chemistry; D004352:Drug Resistance, Microbial; D013293:Enterococcus faecalis / Q000235:genetics; D005434:Flow Cytometry; D015964:Gene Expression Regulation, Bacterial; D005822:Genetic Vectors; D006094:Gram-Positive Bacteria / Q000235:genetics*; D008957:Models, Genetic; D008969:Molecular Sequence Data; D010641:Phenotype; D010957:Plasmids / Q000378:metabolism; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry; D013291:Streptococcus / Q000235:genetics; D014158:Transcription, Genetic
D000900:Anti-Bacterial Agents; D017931:DNA Primers; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D015500:Chloramphenicol O-Acetyltransferase