Experimental determination and characterization of the gap promoter of Bifidobacterium bifidum S17

The DNA sequence upstream of the glyceraldehyde 3-phosphate dehydrogenase gene (gap) of various strains of bifidobacteria is used in a number of vector systems for homologous and heterologous expression in this group of bacteria. To date none of the bifidobacterial gap promoters (P-gap) have been verified experimentally. Here, we probe a range of putative bifidobacterial promoters hypothesized to show high constitutive transcriptional activity using a beta-glucuronidase reporter system. In silico analysis revealed a predicted bacterial promoter upstream of the gap gene of Bifidobacterium bifidum S17. The corresponding DNA sequences was cloned into the promoter probe vector pMDY23 and yielded highest reporter activities among the promoter sequences tested confirming previous studies. Using rapid amplification of cDNA ends (5'-RACE), we identified the transcription start site (TSS) of P-gap of Bifidobacterium. bifidum S17. The experimentally determined TSS and the associated -10 and -35 regions do not match with the promoter predicted in silico. Moreover, a potential ribosome-binding site (RBS) was identified upstream of the ATG start codon of the gap gene, which is complementary to the 3'-end of the 16S rRNA with only 1 mismatch suggesting efficient initiation of translation. Alignment of the P-gap sequences of a number of representative bifidobacteria showed a high level of conservation and the presence of -35 and -10 regions, which are similar but not identical to the consensus promoter sequences of house-keeping genes of Escherichia coli and Bacillus subtilis. Collectively, these results confirm the suitability of P-gap for high level, constitutive expression in bifidobacteria.