An Archaeal Fluoride-Responsive Riboswitch Provides an Inducible Expression System for Hyperthermophiles

Abstract

Robust genetic systems for the hyperthermophilic have facilitated the overexpression of native genes, enabled the addition of sequences encoding secretion signals, epitope, and affinity tags to coding regions, and aided the introduction of sequences encoding new proteins in these fast-growing fermentative heterotrophs. However, tightly controlled and easily manipulated systems facilitating regulated gene expression are limited for these hosts. Here, we describe an alternative method for regulatory control reliant on a-encoded functional riboswitch in the model archaeon Despite the hyperthermophilic growth temperatures, the proposed structure of the riboswitch conforms to a fluoride-responsive riboswitch encoded in many bacteria and similarly functions to regulate a component-conserved fluoride export pathway. Deleting components of the fluoride export pathway generates strains with increased fluoride sensitivity. The mechanism underlying regulated expression suggested that the riboswitch-encoding sequences could be utilized as a tunable expression cassette. When appended to a reporter gene, the riboswitch-mediated control system provides fluoride-dependent tunable regulatory potential, offering an alternative system for regulating gene expression. Riboswitch-regulated expression is thus ubiquitous in extant life and can be exploited to generate regulated expression systems for hyperthermophiles. Gene expression is controlled by a myriad of interconnected mechanisms that interpret metabolic states and environmental cues to balance cell physiology. Transcription regulation in is known to employ both typical repressors-operators and transcription activators to regulate transcription initiation in addition to the regulation afforded by chromatin structure. It was perhaps surprising that the presumed ancient mechanism of riboswitch-mediated regulation is found in and , but seemingly absent in We demonstrate here that a fluoride-responsive riboswitch functions to regulate a detoxification pathway in the hyperthermophilic archaeon The results obtained define a universal role for riboswitch-mediated regulation, adumbrate the presence of several riboswitch-regulated genes in, demonstrate the utility of RNA-based regulation at high temperatures, and provide a novel riboswitch-regulated expression system to employ in hyperthermophiles.

Induction of a Toxin-Antitoxin Gene Cassette under High Hydrostatic Pressure Enables Markerless Gene Disruption in the Hyperthermophilic Archaeon Pyrococcus yayanosii

“…The sugar-inducible system provides a variety of choices for the use of multiple sugars but may interfere with the central metabolism of the organism of interest, which must be taken into consideration. Recently, an archaeal fluoride-responsive riboswitch composed of the Tk-FRR cassette was also constructed as an inducible expression system for hyperthermophiles (31). In our study, we identified an HHP-inducible promoter (P hhp ) in P. yayanosii.…”

FttA is a CPSF73 homologue that terminates transcription in Archaea

“…Given our desire to directly demonstrate that reduced FttA activity impacts termination in vivo , coupled with our inability to generate T. kodakarensis strains encoding enzymatically-impaired FttA variants, we next reduced FttA activity by limiting FttA expression and altering steady-state FttA WT protein levels. To ensure that introduced and regulated expression of TK1428 did not impact TK1429 expression 46 , we separated TK1428 expression from TK1429 by introduction of a new promoter and intrinsic termination sequence, then placed the TK1428 coding sequences downstream of sequences encoding an archaeal fluoride-responsive riboswitch 47 , thereby generating strain IR5 ( Figure 4g ). Construction of IR5 was only possible when cultures were continuously provided with fluoride even though fluoride impairs growth of T. kodakarensis 47 , supporting that very limited expression of TK1428 was not compatible with life.…”

“…To ensure that introduced and regulated expression of TK1428 did not impact TK1429 expression 46 , we separated TK1428 expression from TK1429 by introduction of a new promoter and intrinsic termination sequence, then placed the TK1428 coding sequences downstream of sequences encoding an archaeal fluoride-responsive riboswitch 47 , thereby generating strain IR5 ( Figure 4g ). Construction of IR5 was only possible when cultures were continuously provided with fluoride even though fluoride impairs growth of T. kodakarensis 47 , supporting that very limited expression of TK1428 was not compatible with life. Steady-state FttA levels in IR5 strains grown in the absence and presence of fluoride reveals a modest ~2-fold change in FttA levels in vivo when fluoride is removed from cultures ( Figure 4h and Supplementary Information Figure 2 ), yet even this modest alteration significantly and reproducibly impacts transcription termination in vivo ( Figure 4d - f ).…”

Comprehensive sequence-to-function mapping of cofactor-dependent RNA catalysis in the glmS ribozyme

“…A cross all three domains of life, evolution has generated a diverse set of functional RNAs known to play important roles in cellular homeostasis and gene regulation [1][2][3][4][5][6] . These biopolymers fold into modular, three-dimensional structures capable of binding specific ligands (aptamers) or catalyzing specific biochemical reactions (ribozymes).…”