Controlling Bacterial Transcription with an Archaeal Transcription Regulator

During the bottom-up engineering of synthetic genetic circuits, it is essential to minimize cross-talk with the host metabolism and to attain orthogonality. In this context, it is a promising approach to leverage gene regulatory parts from across the borders of the bacterial and archaeal domains of life. Archaea harbour a simplified version of the eukaryotic RNA polymerase II machinery, with a 13-subunit RNA polymerase and additional basal transcription factors TATA binding protein and transcription factor B. In contrast, bacterial and archaeal regulatory transcription factors are similar and typically harbour two domains of which one helix-turn-helix DNA-binding domain. In this work, we aim to build an orthogonal genetic circuit by introducing an archaeal transcription factor, the Lrp-type beta-alanine-responsive activator BarR from Sulfolobus acidocaldarius, in a bacterial system.

Two compatible plasmids were introduced into E. coli, one harbouring a barR gene under control of an IPTG-inducible promoter and one harbouring promoter/operator sequences fused to a fluorescent reporter gene. Upon combining the natural archaeal barR promoter/operator region to the reporter gene, expression was observed indicating that despite the fundamental difference between the respective transcription machineries, the bacterial RNA polymerase recognizes and initiates transcription from an archaeal promoter region. Moreover, upon inducing BarR expression, a strong repression was observed, presumably due to steric promoter occlusion. Next, we performed similar experiments using plasmid constructs in which different versions of the BarR operator were fused to a bacterial promoter and the reporter gene. This revealed that fusion constructs harbouring a single operator site displayed a slight BarR-dependent transcriptional activation, but only in the stationary growth phase. This activation was not observed anymore when cells were grown in the presence of beta-alanine. Altogether, this work demonstrates that prokaryotic transcription factors have a flexible nature and are capable of interacting with both bacterial and archaeal transcription machineries, in a positive or negative manner.