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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.    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.

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