TPP riboswitch characterization in Alishewanella tabrizica and Alishewanella aestuarii and comparison with other TPP riboswitches

Riboswitches are located in non-coding areas of mRNAs and act as sensors of cellular small molecules, regulating gene expression in response to ligand binding. The TPP riboswitch is the most widespread riboswitch occurring in all three domains of life. However, it has been rarely characterized in environmental bacteria other than Escherichia coli and Bacillus subtilis. In this study, TPP riboswitches located in the 5′ UTR of thiC operon from Alishewanella tabrizica and Alishewanella aestuarii were identified and characterized. Moreover, affinity analysis of TPP binding to the TPP aptamer domains originated from A. tabrizica, A. aestuarii, E.coli, and B. subtilis were studied and compared using In-line probing and Surface Plasmon Resonance (SPR). TPP binding to the studied RNAs from A. tabrizica and A. aestuarii caused distinctive changes of the In-line cleavage pattern, demonstrating them as functional TPP riboswitches. With dissociation constant of 2-4 nM (depending on the method utilized), the affinity of TPP binding was highest in A. tabrizica, followed by the motifs sourced from A. aestuarii, E. coli, and B. subtilis. The observed variation in their TPP-binding affinity might be associated with adaptation to the different environments of the studied bacteria.