Experimental determination and characterization of the gap promoter of XXXX .

@entity1021 @entity16335 @entity60900 @entity3881 @entity3337 @entity526
The DNA sequence upstream of the @entity16335 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 (Pgap) have been verified experimentally. Here, we probe a range of putative bifidobacterial promoters hypothesized to show high constitutive transcriptional activity using a b-glucuronidase reporter system. In silico analysis revealed a predicted bacterial promoter upstream of the gap gene of @entity60900 . 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 @entity1021 ( @entity1021 ) of Pgap of @entity60900 . The experimentally determined @entity1021 and the associated -10 and -35 regions do not match with the promoter predicted in silico. Moreover, a potential @entity3337 ( @entity3337 ) 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 Pgap 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 @entity526 and @entity3881 . Collectively, these results confirm the suitability of Pgap for high level, constitutive expression in bifidobacteria.
