Light-Dependent Control of Bacterial Expression at the mRNA Level

Abstract

Sensory photoreceptors mediate numerous light-dependent adaptations across organisms. In optogenetics, photoreceptors achieve the reversible, non-invasive, and spatiotemporally precise control by light of gene expression and other cellular processes. The light-oxygen-voltage receptor PAL binds to small RNA aptamers with sequence specificity upon blue-light illumination. By embedding the responsive aptamer in the ribosome-binding sequence of genes of interest, their expression can be downregulated by light. We developed the pCrepusculo and pAurora optogenetic systems that are based on PAL and allow to down-and upregulate, respectively, bacterial gene expression using blue light. Both systems are realized as compact, single plasmids that exhibit stringent blue-light responses with low basal activity and up to several 10-fold dynamic range. As PAL exerts light-dependent control at the RNA level, it can be combined with other optogenetic circuits that control transcription initiation. By integrating regulatory mechanisms operating at the DNA and mRNA levels, optogenetic circuits with emergent properties can thus be devised. As a case in point, the pEnumbra setup permits to upregulate gene expression under moderate blue light whereas strong blue light shuts off expression again. Beyond providing novel signal-responsive expression systems for diverse applications in biotechnology and synthetic biology, our work also illustrates how the light-dependent PAL-aptamer interaction can be harnessed for the control and interrogation of RNA-based processes.

Light-regulated gene expression in Bacteria: Fundamentals, advances, and perspectives

“…The latter consideration certainly holds true for light-regulated gene-expression systems which in most cases realize regulation at the transcriptional level, see control points for optogenetic regulation below. Although the experimental data on this aspect are sparse, several optogenetic setups for light-regulated expression in bacteria exhibited significant changes in expression levels within around half an hour after light exposure, with the response taking several hours to manifest to full degree (Ohlendorf et al, 2012;Olson et al, 2014Olson et al, , 2017Ramakrishnan and Tabor, 2016;Multamäki et al, 2022;Ranzani et al, 2022). The dark-recovery kinetics greatly differ across photoreceptor families and their individual members.…”

“…Binding to the 5′-UTR may mask the ribosome-binding site (or, Shine-Dalgarno [SD] sequence) and thereby interfere with translation initiation (Ranzani et al, 2022). Alternatively, binding to the 3′-UTR could be harnessed for regulating intracellular RNA stability (Tang and Guest, 1999).…”

“…(E) The LOV receptor NmPAL from N. multipartita binds specific RNA sequences upon blue-light activation. Once embedded adjacent to the Shine-Dalgarno sequence (SD) of target mRNAs, light-induced PAL binding to these sequences interferes with ribosome binding and reduces expression (Weber et al, 2019;Ranzani et al, 2022).…”

“…In darkness, the ANTAR moiety of PAL is in tight complex with a LOV module and thus autoinhibited. Blue light relieves the intramolecular inhibition and thereby increases the affinity of PAL for specific RNA hairpins, denoted aptamers in the following, by 100-fold or more to between 5 and 20 nM, depending on RNA sequence (Weber et al, 2019;Ranzani et al, 2022). The sequence-specific, lightactivated RNA binding of PAL was leveraged for the regulation of bacterial expression by interleaving the aptamer with the Shine-Dalgarno (SD) sequence of a target gene (Figure 4E).…”

“…Once activated by light, PAL could then bind to this region of the mRNA and thereby interfere with translation, which led to an up to 10-fold reduction of fluorescent reporter expression at 29 °C (Weber et al, 2019). To facilitate the adoption of PALbased optoribogenetic circuits, we recently developed the pCrepusculo system which is realized on a single plasmid and implements an improved aptamer sequence with higher affinity for PAL (Ranzani et al, 2022). pCrepusculo enabled the 8-fold downregulation of expression at 37 °C.…”