Theophylline-responsive Riboswitch Research Articles

Molecular Dynamics Simulations of Ribosome-Binding Sites in Theophylline-Responsive Riboswitch Associated with Improving the gene Expression Regulation in Chloroplasts

Molecular Dynamics Simulations of Ribosome-Binding Sites in Theophylline-Responsive Riboswitch Associated with Improving the gene Expression Regulation in Chloroplasts

Use of CRISPR interference for efficient and rapid gene inactivation in Fusobacterium nucleatum.

Gene inactivation by creating in-frame deletion mutations in Fusobacterium nucleatum is time consuming, and most fusobacterial strains are genetically intractable. Addressing these problems, we introduced a riboswitch-based inducible CRISPR interference (CRISPRi) system. This system employs the nuclease-inactive Streptococcus pyogenes Cas9 protein (dCas9), specifically guided to the gene of interest by a constantly expressed single-guide RNA (sgRNA). Mechanistically, this dCas9-sgRNA complex serves as an insurmountable roadblock for RNA polymerase, thus repressing the target gene transcription. Leveraging this system, we first examined two non-essential genes, ftsX and radD, which are pivotal for fusobacterial cytokinesis and coaggregation. Upon adding the inducer, theophylline, ftsX suppression caused filamentous cell formation akin to chromosomal ftsX deletion, while targeting radD significantly reduced RadD protein levels, abolishing RadD-mediated coaggregation. The system was then extended to probe essential genes bamA and ftsZ, which are vital for outer membrane biogenesis and cell division. Impressively, bamA suppression disrupted membrane integrity and bacterial separation, stalling growth, while ftsZ targeting yielded elongated cells in broth with compromised agar growth. Further studies on F. nucleatum clinical strain CTI-2 and Fusobacterium periodonticum revealed reduced indole synthesis when targeting tnaA. Moreover, silencing clpB in F. periodonticum decreased ClpB, increasing thermal sensitivity. In summary, our CRISPRi system streamlines gene inactivation across various fusobacterial strains.IMPORTANCEHow can we effectively investigate the gene functions in Fusobacterium nucleatum, given the dual challenges of gene inactivation and the inherent genetic resistance of many strains? Traditional methods have been cumbersome and often inadequate. Addressing this, our work introduces a novel inducible CRISPR interference (CRISPRi) system in which dCas9 expression is controlled at the translation level by a theophylline-responsive riboswitch unit, and single-guide RNA expression is driven by the robust, constitutive rpsJ promoter. This approach simplifies gene inactivation in the model organism (ATCC 23726) and extends its application to previously considered genetically intractable strains like CTI-2 and Fusobacterium periodonticum. With CRISPRi's potential, it is a pivotal tool for in-depth genetic studies into fusobacterial pathogenesis, potentially unlocking targeted therapeutic strategies.

Development of a xylose-inducible promoter and riboswitch combination system for manipulating gene expression in Fusobacterium nucleatum.

Inducible gene expression systems are important for studying bacterial gene function, yet most exhibit leakage. In this study, we engineered a leakage-free hybrid system for precise gene expression controls in Fusobacterium nucleatum by integrating the xylose-inducible expression system with the theophylline-responsive riboswitch. This innovative method enables concurrent control of target gene expression at both transcription and translation initiation levels. Using luciferase and the indole-producing enzyme tryptophanase (TnaA) as reporters, we demonstrated that the hybrid system displays virtually no observable signal in the absence of inducers. We employed this system to express FtsX, a protein related to fusobacterial cytokinesis, in an ftsX mutant strain, unveiling a dose-dependent manner in FtsX production. Without inducers, cells form long filaments, while increasing FtsX levels by increasing inducer concentrations led to a gradual reduction in cell length until normal morphology was restored. Crucially, this system facilitated essential gene investigation, identifying the signal peptidase lepB gene as vital for F. nucleatum. LepB's essentiality stems from depletion, affecting outer membrane biogenesis and cell division. This novel hybrid system holds the potential for advancing research on essential genes and accurate gene regulation in F. nucleatum. IMPORTANCE Fusobacterium nucleatum, an anaerobic bacterium prevalent in the human oral cavity, is strongly linked to periodontitis and can colonize areas beyond the oral cavity, such as the placenta and gastrointestinal tract, causing adverse pregnancy outcomes and promoting colorectal cancer growth. Given F. nucleatum's clinical significance, research is underway to develop targeted therapies to inhibit its growth or eradicate the bacterium specifically. Essential genes, crucial for bacterial survival, growth, and reproduction, are promising drug targets. A leak-free-inducible gene expression system is needed for studying these genes, enabling conditional gene knockouts and elucidating the importance of those essential genes. Our study identified lepB as the essential gene by first generating a conditional gene mutation in F. nucleatum. Combining a xylose-inducible system with a riboswitch facilitated the analysis of essential genes in F. nucleatum, paving the way for potential drug development targeting this bacterium for various clinical applications.

HicA Toxin-Based Counterselection Marker for Allelic Exchange Mutations in Fusobacterium nucleatum.

The study of fusobacterial virulence factors has dramatically benefited from the creation of various genetic tools for DNA manipulation, including galK-based counterselection for in-frame deletion mutagenesis in Fusobacterium nucleatum, which was recently developed. However, this method requires a host lacking the galK gene, which is an inherent limitation. To circumvent this limitation, we explored the possibility of using the hicA gene that encodes a toxin consisting of a HicAB toxin-antitoxin module in Fusobacterium periodonticum as a new counterselective marker. Interestingly, the full-length hicA gene is not toxic in F. nucleatum, but a truncated hicA gene version lacking the first six amino acids is functional as a toxin. The toxin expression is driven by an rpsJ promoter and is controlled at its translational level by using a theophylline-responsive riboswitch unit. As a proof of concept, we created markerless in-frame deletions in the fusobacterial adhesin radD gene within the F. nucleatum rad operon and the tnaA gene that encodes the tryptophanase for indole production. After vector integration, plasmid excision after counterselection appeared to have occurred in 100% of colonies grown on theophylline-added plates and resulted in in-frame deletions in 50% of the screened isolates. This hicA-based counterselection system provides a robust and reliable counterselection in wild-type background F. nucleatum and should also be adapted for use in other bacteria. IMPORTANCE Fusobacterium nucleatum is an indole-producing human oral anaerobe associated with periodontal diseases, preterm birth, and several cancers. Little is known about the mechanisms of fusobacterial pathogenesis and associated factors, mainly due to the lack of robust genetic tools for this organism. Here, we showed that a mutated hicA gene from Fusobacterium periodonticum expresses an active toxin and was used as a counterselection marker. This hicA-based in-frame deletion system efficiently creates in-frame deletion mutations in the wild-type background of F. nucleatum. This is the first report to use the hicA gene as a counterselection marker in a bacterial genetic study.

Sustainable citric acid production from CO2 in an engineered cyanobacterium.

Citric acid is one of the most widely used organic acids in the world, with applications ranging from acidity regulation in food and beverages to metal chelation in hydrometallurgical processes. Most of its production is currently derived from fermentative processes, using plant-derived carbon feedstocks. While these are currently dominant, there is an increasing need to develop closed-loop production systems that reduce process carbon footprint. In this work, we demonstrate for the first time that an engineered marine cyanobacterium Synechococcus sp. PCC 7002 can be used as a sustainable chassis for the photosynthetic conversion of CO2 to citric acid. Decreased citric acid cycle flux, through the use of a theophylline-responsive riboswitch, was combined with improved flux through citrate synthase and enhanced citric acid excretion, resulting in a significant improvement to citric acid production. While allowing citrate production, this strategy induces a growth defect which can be overcome by glutamate supplementation or by fine-tuning aconitase levels, resulting in an increase in production relative to WT of over 100-fold. This work represents a first step toward sustainable production of a commodity organic acid from CO2.

Inducible CRISPR/Cas9 Allows for Multiplexed and Rapidly Segregated Single-Target Genome Editing in Synechocystis Sp. PCC 6803.

Establishing various synthetic biology tools is crucial for the development of cyanobacteria for biotechnology use, especially tools that allow for precise and markerless genome editing in a time-efficient manner. Here, we describe a riboswitch-inducible CRISPR/Cas9 system, contained on a single replicative vector, for the model cyanobacterium Synechocystis sp. PCC 6803. A theophylline-responsive riboswitch allowed tight control of Cas9 expression, which enabled reliable transformation of the CRISPR/Cas9 vector intoSynechocystis. Induction of the CRISPR/Cas9 mediated various types of genomic edits, specifically deletions and insertions of varying size. The editing efficiency varied depending on the target and intended edit; smaller edits performed better, reaching, e.g., 100% for insertion of a FLAG-tag onto rbcL. Importantly, the single-vector CRISPR/Cas9 system mediated multiplexed editing of up to three targets in parallel inSynechocystis. All single-target and several double-target mutants were also fully segregated after the first round of induction. Lastly, a vector curing system based on the nickel-inducible expression of the toxic mazF (from Escherichia coli) was added to the CRISPR/Cas9 vector. This inducible system allowed for curing of the vector in 25–75% of screened colonies, enabling edited mutants to become markerless.

A CRISPR-Based Method for Constructing Conditional Mutations of Essential Genes in Cyanobacteria.

Cyanobacteria, a group of diverse bacteria capable of oxygenic photosynthesis, are excellent models for investigating many important cellular processes, such as photosynthesis, nitrogen fixation, and prokaryotic cell differentiation. They also have great potential to become the next-generation cell factories for sustainable biosynthesis of valuable products. However, genetic manipulation in cyanobacteria is not as convenient as in other model bacteria. Particularly, handling essential genes in cyanobacteria has been difficult due to the lack of appropriate tools, limiting our understanding of many important cellular functions encoded by them. We recently develop a CRISPR-based method for constructing the conditional mutants of cyanobacterial essential genes by engineering the ribosome binding site to a theophylline-responsive riboswitch. Here, we provide the details of this method. The principle of this method could be used to construct conditional mutants in a wide range of bacterial species.

A modular chromosomally integrated toolkit for ectopic gene expression in Vibrio cholerae

The ability to express genes ectopically in bacteria is essential for diverse academic and industrial applications. Two major considerations when utilizing regulated promoter systems for ectopic gene expression are (1) the ability to titrate gene expression by addition of an exogenous inducer and (2) the leakiness of the promoter element in the absence of the inducer. Here, we describe a modular chromosomally integrated platform for ectopic gene expression in Vibrio cholerae. We compare the broadly used promoter elements Ptac and PBAD to versions that have an additional theophylline-responsive riboswitch (Ptac-riboswitch and PBAD-riboswitch). These constructs all exhibited unimodal titratable induction of gene expression, however, max induction varied with Ptac > PBAD > PBAD-riboswitch > Ptac-riboswitch. We also developed a sensitive reporter system to quantify promoter leakiness and show that leakiness for Ptac > Ptac-riboswitch > PBAD; while the newly developed PBAD-riboswitch exhibited no detectable leakiness. We demonstrate the utility of the tightly inducible PBAD-riboswitch construct using the dynamic activity of type IV competence pili in V. cholerae as a model system. The modular chromosomally integrated toolkit for ectopic gene expression described here should be valuable for the genetic study of V. cholerae and could be adapted for use in other species.

A Reversibly Induced CRISPRi System Targeting Photosystem II in the Cyanobacterium Synechocystis sp. PCC 6803.

The cyanobacterium Synechocystis sp. PCC 6803 is used as a model organism to study photosynthesis, as it can utilize glucose as the sole carbon source to support its growth under heterotrophic conditions. CRISPR interference (CRISPRi) has been widely applied to repress the transcription of genes in a targeted manner in cyanobacteria. However, a robust and reversible induced CRISPRi system has not been explored in Synechocystis 6803 to knock down and recover the expression of a targeted gene. In this study, we built a tightly controlled chimeric promoter, P rhaBAD-RSW, in which a theophylline responsive riboswitch was integrated into a rhamnose-inducible promoter system. We applied this promoter to drive the expression of ddCpf1 (DNase-dead Cpf1 nuclease) in a CRISPRi system and chose the PSII reaction center gene psbD (D2 protein) to target for repression. psbD was specifically knocked down by over 95% of its native expression, leading to severely inhibited photosystem II activity and growth of Synechocystis 6803 under photoautotrophic conditions. Significantly, removal of the inducers rhamnose and theophylline reversed repression by CRISPRi. Expression of PsbD recovered following release of repression, coupled with increased photosystem II content and activity. This reversibly induced CRISPRi system in Synechocystis 6803 represents a new strategy for study of the biogenesis of photosynthetic complexes in cyanobacteria.

A Theophylline-Responsive Riboswitch Regulates Expression of Nuclear-Encoded Genes.

Riboswitches are small cis-regulatory RNA elements that regulate gene expression by conformational changes in response to ligand binding. Synthetic riboswitches have been engineered as versatile and innovative tools for gene regulation by external application of their ligand in prokaryotes and eukaryotes. In plants, synthetic riboswitches were used to regulate gene expression in plastids, but the application of synthetic riboswitches for the regulation of nuclear-encoded genes in planta remains to be explored. Here, we characterize the properties of a theophylline-responsive synthetic aptazyme for control of nuclear-encoded transgenes in Arabidopsis (Arabidopsis thaliana). Activation of the aptazyme, inserted in the 3' UTR of the target gene, resulted in rapid self-cleavage and subsequent decay of the mRNA. This riboswitch allowed reversible, theophylline-dependent down-regulation of the GFP reporter gene in a dose- and time-dependent manner. Insertion of the riboswitch into the ONE HELIX PROTEIN1 gene allowed complementation of ohp1 mutants and induction of the mutant phenotype by theophylline. GFP and ONE HELIX PROTEIN1 transcript levels were downregulated by up to 90%, and GFP protein levels by 95%. These results establish artificial riboswitches as tools for externally controlled gene expression in synthetic biology in plants or functional crop design.

Adopting a Theophylline-Responsive Riboswitch for Flexible Regulation and Understanding of Glycogen Metabolism in Synechococcus elongatus PCC7942.

Cyanobacteria are supposed to be promising photosynthetic microbial platforms that recycle carbon dioxide driven into biomass and bioproducts by solar energy. Glycogen synthesis serves as an essential natural carbon sink mechanism, storing a large portion of energy and organic carbon source of photosynthesis. Engineering glycogen metabolism to harness and rewire carbon flow is an important strategy to optimize efficacy of cyanobacteria platforms. ADP-glucose pyrophosphorylase (GlgC) catalyzes the rate-limiting step for glycogen synthesis. However, knockout of glgC fails to promote cell growth or photosynthetic production in cyanobacteria, on the contrary, glgC deficiency impairs cellular fitness and robustness. In this work, we adopted a theophylline-responsive riboswitch to engineer and control glgC expression in Synechococcus elongatus PCC7942 and achieved flexible regulation of intracellular GlgC abundance and glycogen storage. With this approach, glycogen synthesis and glycogen contents in PCC7942 cells could be regulated in a range from about 40 to 300% of wild type levels. In addition, the results supported a positive role of glycogen metabolism in cyanobacteria cellular robustness. When glycogen storage was reduced, cellular physiology and growth under standard conditions was not impaired, while cellular tolerance toward environmental stresses was weakened. While when glycogen synthesis was enhanced, cells of PCC7942 displayed optimized cellular robustness. Our findings emphasize the significance of glycogen metabolism for cyanobacterial physiology and the importance of flexible approaches for engineering and understanding cellular physiology and metabolism.

A tightly inducible riboswitch system in Synechocystis sp. PCC 6803.

Cyanobacteria are photosynthetic microorganisms that serve as experimental model organisms for the study of photosynthesis, environmental stress responses, and the production of biofuels. Genetic tools for bioengineering have been developed as a result of such studies. However, there is still room for improvement for the tight control of experimental protein expression in these microorganisms. Here, we describe an expression system controlled by a theophylline-responsive riboswitch that we have constructed in the cyanobacterium Synechocystis sp. PCC 6803. We demonstrate that, in response to different theophylline concentrations, this riboswitch can tightly control green fluorescence protein expression in Synechocystis. Thus, this system is useful as a tool for genetic engineering and the synthetic biology of cyanobacteria.

Use of a Riboswitch-controlled Conditional Hypomorphic Mutation to Uncover a Role for the Essential csrA Gene in Bacterial Autoaggregation

Essential genes encode biological functions critical for cell survival. Correspondingly, their null mutants are often difficult to obtain, which impedes subsequent genetic and functional analysis. Here, we describe the development and utility of a theophylline-responsive riboswitch that enables target gene expression to be specifically "tuned" from low to high levels, which may be used to generate conditional hypomorphic mutants. Low levels of gene activity in the absence of the ligand (theophylline) permit cell survival, enabling gene activities to be investigated. Normal gene expression levels and wild-type phenotypes can be restored by the addition of the ligand. We demonstrate the utility of this approach with csrA, an essential gene in Escherichia coli that encodes the global regulatory protein CsrA. We placed the theophylline-responsive riboswitch immediately upstream of the csrA ribosome binding site, with the resulting mutant named switch-csrA. Hypomorphism of switch-csrA and its specific responsiveness to theophylline were verified by phenotypic examination and translation analysis. The utility of switch-csrA revealed a previously unidentified function for CsrA, namely its role as a repressor of cellular autoaggregation. Specifically, switch-csrA in the non-ligand-bound form produced low levels of CsrA, and its cells autoaggregated. Theophylline binding induced conformational changes in the riboswitch and permitted efficient csrA translation; consequently, autoaggregation did not occur. Our results indicate that CsrA modulates autoaggregation via the polysaccharide adhesin poly-beta-1,6-N-acetyl-D-glucosamine. In summary, the use of ligand-responsive riboswitches to construct conditional hypomorphic mutants represents a novel approach for investigating the activities of essential genes, which effectively complements traditional genetic approaches.

Sequestering of the 3′ Splice Site in a Theophylline-Responsive Riboswitch Allows Ligand-Dependent Control of Alternative Splicing

Despite the important role of alternative splicing in various aspects of biological processes, our ability to regulate this process at will remains a challenge. In this report, we asked whether a theophylline-responsive riboswitch could be adapted to manipulate alternative splicing. We constructed a pre-mRNA containing a single upstream 5' splice site and two 3' splice sites, of which the proximal 3' splice site is embedded in theophylline-responsive riboswitch. We show that this pre-mRNA spliced with preferential utilization of proximal 3' splice site in vitro. However, addition of theophylline to the splicing reaction promoted splicing at distal 3' splice site thereby changing the ratio of distal-to-proximal 3' splice site usage by more than twofold. Our data suggest that theophylline influenced 3' splice site choice without affecting the kinetics of the splicing reaction. We conclude that an in vitro selected riboswitch can be adapted to control alternative splicing, which may find many applications in basic, biotechnological, and biomedical research.