Characterization of the structure and function of a Bacteroides thetaiotaomicron 16S rRNA promoter
Thorson, Mary Leah
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The bacteroides group is a subdivision in the Cytophaga-Flavobacterium-Bacteroides phylum. This group is as phylogenetically distinct from other Gram-negative enterics, including Escherichia coli, as they are from Gram-positive organisms. Furthermore, there is no cross expression between genes of E. coli and Bacteroides species. It is thought that this difference in gene expression lies in part at the level of transcription initiation and is due to the sequences within the promoter region itself. A putative consensus sequence for Bacteroides promoters has been published by C. Jeff Smiths research group based on alignments of the sequences upstream of certain regulated genes. However, this consensus has not been found within all putative Bacteroides promoters. In this study, the promoter structure and function of a strong housekeeping B. thetaiotaomicron 16S rRNA promoter was examined and compared to an E. coli 16S rRNA promoter. Our hypothesis is that there are significant differences between the promoters of these two organisms. Analysis of B. thetaiotaomicron sequence upstream of the 16S rRNA gene has revealed the same overall structure known for E. coli 16S rRNA promoters in that there are two putative promoters separated by approximately 150 bp. However, the B. thetaiotaomicron 16S rRNA promoter contains the proposed Bacteroides 7 and 33 consensus sequences instead of the well known E. coli 10 and 35 consensus sequences. The biological activity of the B. thetaiotaomicron 16S rRNA full-length promoter was confirmed using a Bacteroides lux reporter system. A newly designed Bacteroides lux reporter was used to analyze specific regions of the B. thetaiotaomicron 16S rRNA promoter. In addition, by pairing the B. thetaiotaomicron 16S rRNA promoter with an E. coli ribosomal binding site, and vice-versa, the improved lux reporter was used to further confirm that the difference in gene expression between the two species lies at the level of transcription in E. coli. In Bacteroides, however, transcription and translation may work together to create a barrier to efficient gene expression of foreign genes.
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