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The C repressor protein of phage 16-3, which is required for establishing and maintaining lysogeny, recognizes structurally different operators which differ by 2 bp in the length of the spacer between the conserved palindromic sequences. A 'rotationally flexible protein homodimers' model has been proposed in order to explain the conformational adaptivity of the 16-3 repressor. In this paper, we report on the isolation of a repressor mutant with altered binding specificity which was used to identify a residue-base pair contact and to monitor the spatial relationship of the recognition helix of C repressor to the contacting major groove of DNA within the two kinds of repressor-operator complexes. Our results indicate spatial differences at the interface which may reflect different docking arrangements in recognition of the structurally different operators by the 16-3 repressor.

We developed an orally administered, engineered, bacterium-based, RNA interference-mediated therapeutic method to significantly reduce the symptoms in the most frequently used animal model of inflammatory bowel disease. This bacterium-mediated RNA interference strategy was based on the genomically stable, non-pathogenic E. coli MDS42 strain, which was engineered to constitutively produce invasin and the listeriolysin O cytolysin. These proteins enabled the bacteria first to invade the colon epithelium and then degrade in the phagosome. This allowed the delivery of a plasmid encoding small hairpin RNA (shRNA) targeting tumor necrosis factor (TNF) into the cytoplasm of the target cells. The expression levels of TNF and other cytokines significantly decreased upon this treatment in dextran sulfate sodium (DSS)-induced colitis, and the degree of inflammation was significantly reduced. With further safety modifications this method could serve as a safe and side effect-free alternative to biologicals targeting TNF or other inflammatory mediators., Competing Interests: The authors declare no competing interests., (© 2020 The Author(s).)