Modular riboswitch toolsets for synthetic genetic control in diverse bacterial species.

Ligand-dependent control of gene expression is essential for gene functional analysis, target validation, protein production, and metabolic engineering. However, the expression tools currently available are difficult to transfer between species and exhibit limited mechanistic diversity. Here we demonstrate how the modular architecture of purine riboswitches can be exploited to develop orthogonal and chimeric switches that are transferable across diverse bacterial species, modulating either transcription or translation, to provide tunable activation or repression of target gene expression, in response to synthetic non-natural effector molecules. Our novel riboswitch-ligand pairings are shown to regulate physiologically important genes required for bacterial motility in Escherichia coli and cell morphology in Bacillus subtilis. These findings are relevant for future gene function studies and antimicrobial target validation, while providing new modular and orthogonal regulatory components for deployment in synthetic biology regimes.

[1]  R. Montange,et al.  Structure of a natural guanine-responsive riboswitch complexed with the metabolite hypoxanthine , 2004, Nature.

[2]  Randy J. Read,et al.  Phaser crystallographic software , 2007, Journal of applied crystallography.

[3]  Andrea L Edwards,et al.  Adaptive ligand binding by the purine riboswitch in the recognition of guanine and adenine analogs. , 2009, Structure.

[4]  Jeffrey E. Barrick,et al.  Tandem Riboswitch Architectures Exhibit Complex Gene Control Functions , 2006, Science.

[5]  R. Batey,et al.  Modified pyrimidines specifically bind the purine riboswitch. , 2006, Journal of the American Chemical Society.

[6]  Yohei Yokobayashi,et al.  Engineering complex riboswitch regulation by dual genetic selection. , 2008, Journal of the American Chemical Society.

[7]  Jeffrey H. Miller.  Experiments in molecular genetics , 1972 .

[8]  Beatrix Suess,et al.  Engineered riboswitches: Expanding researchers' toolbox with synthetic RNA regulators , 2012, FEBS letters.

[9]  D. Metzgar,et al.  Identification of Four Genes Necessary for Biosynthesis of the Modified Nucleoside Queuosine* , 2004, Journal of Biological Chemistry.

[10]  E. Freire,et al.  Thermodynamic mapping of the inhibitor site of the aspartic protease endothiapepsin. , 1995, Journal of molecular biology.

[11]  Harald Schwalbe,et al.  Three-state mechanism couples ligand and temperature sensing in riboswitches , 2013, Nature.

[12]  Andrea L Edwards,et al.  Riboswitches: structures and mechanisms. , 2011, Cold Spring Harbor perspectives in biology.

[13]  Markus Wieland,et al.  Artificial ribozyme switches containing natural riboswitch aptamer domains. , 2009, Angewandte Chemie.

[14]  Jay D Keasling,et al.  Effect of lacYExpression on Homogeneity of Induction from the Ptac and Ptrc Promoters by Natural and Synthetic Inducers , 2002, Biotechnology progress.

[15]  J. Cronan,et al.  Long-term and homogeneous regulation of the Escherichia coli araBAD promoter by use of a lactose transporter of relaxed specificity , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[16]  Kevin Cowtan,et al.  research papers Acta Crystallographica Section D Biological , 2005 .

[17]  H. Fukada,et al.  Enthalpy and heat capacity changes for the proton dissociation of various buffer components in 0.1 M potassium chloride , 1998, Proteins.

[18]  A. Serganov,et al.  A Decade of Riboswitches , 2013, Cell.

[19]  R. Batey,et al.  Modularity of select riboswitch expression platforms enables facile engineering of novel genetic regulatory devices. , 2013, ACS synthetic biology.

[20]  K. P. Murphy,et al.  Evaluation of linked protonation effects in protein binding reactions using isothermal titration calorimetry. , 1996, Biophysical journal.

[21]  Eric D Brown,et al.  A FACS‐Based Approach to Engineering Artificial Riboswitches , 2008, Chembiochem : a European journal of chemical biology.

[22]  Randy J Read,et al.  Electronic Reprint Biological Crystallography Phenix: Building New Software for Automated Crystallographic Structure Determination Biological Crystallography Phenix: Building New Software for Automated Crystallographic Structure Determination , 2022 .

[23]  Jun Wang,et al.  Platensimycin is a selective FabF inhibitor with potent antibiotic properties , 2006, Nature.

[24]  A. Serganov,et al.  Structural basis for discriminative regulation of gene expression by adenine- and guanine-sensing mRNAs. , 2004, Chemistry & biology.

[25]  X. Zhuang,et al.  Coupled, Circumferential Motions of the Cell Wall Synthesis Machinery and MreB Filaments in B. subtilis , 2011, Science.

[26]  B. Nall,et al.  Enthalpy of antibody--cytochrome c binding. , 1995, Biochemistry.

[27]  D. Sept,et al.  A22 disrupts the bacterial actin cytoskeleton by directly binding and inducing a low-affinity state in MreB. , 2009, Biochemistry.

[28]  Judith P. Armitage,et al.  Signal processing in complex chemotaxis pathways , 2011, Nature Reviews Microbiology.

[29]  Jeffrey E. Barrick,et al.  Riboswitches Control Fundamental Biochemical Pathways in Bacillus subtilis and Other Bacteria , 2003, Cell.

[30]  Wolfgang Kabsch,et al.  Automatic processing of rotation diffraction data from crystals of initially unknown symmetry and cell constants , 1993 .

[31]  Michael Famulok,et al.  Aptamers for allosteric regulation. , 2011, Nature chemical biology.

[32]  J. Gallivan,et al.  A flow cytometry-based screen for synthetic riboswitches , 2008, Nucleic acids research.

[33]  Yohei Yokobayashi,et al.  Controlling Mammalian Gene Expression by Allosteric Hepatitis Delta Virus Ribozymes , 2013, ACS synthetic biology.

[34]  T. Harris,et al.  Thermodynamically balanced inside-out (TBIO) PCR-based gene synthesis: a novel method of primer design for high-fidelity assembly of longer gene sequences. , 2003, Nucleic acids research.

[35]  J. Micklefield,et al.  Reengineering orthogonally selective riboswitches , 2010, Proceedings of the National Academy of Sciences.

[36]  D. Sherman,et al.  Pathway-selective sensitization of Mycobacterium tuberculosis for target-based whole-cell screening. , 2012, Chemistry & biology.

[37]  B. Golden,et al.  Direct measurement of a pK(a) near neutrality for the catalytic cytosine in the genomic HDV ribozyme using Raman crystallography. , 2007, Journal of the American Chemical Society.

[38]  Jason Micklefield,et al.  Orthogonal riboswitches for tuneable coexpression in bacteria. , 2012, Angewandte Chemie.

[39]  Benedikt Klauser,et al.  In vivo screening of ligand-dependent hammerhead ribozymes. , 2012, Methods in molecular biology.

[40]  Shana Topp,et al.  Random Walks to Synthetic Riboswitches—A High‐Throughput Selection Based on Cell Motility , 2008, Chembiochem : a European journal of chemical biology.

[41]  J. Gallivan,et al.  Guiding bacteria with small molecules and RNA. , 2007, Journal of the American Chemical Society.

[42]  J. Sturtevant,et al.  Calorimetric studies of the activation of chymotrypsinogen A. , 1971, Biochemistry.

[43]  P. Stragier,et al.  Plasmids for ectopic integration in Bacillus subtilis. , 1996, Gene.

[44]  J. Errington,et al.  A magnesium‐dependent mreB null mutant: implications for the role of mreB in Bacillus subtilis , 2005, Molecular microbiology.

[45]  R. Breaker.  Riboswitches and the RNA world. , 2012, Cold Spring Harbor perspectives in biology.

[46]  V. Fromion,et al.  Processive Movement of MreB-Associated Cell Wall Biosynthetic Complexes in Bacteria , 2011, Science.

[47]  Robert T. Batey,et al.  Engineering modular ‘ON’ RNA switches using biological components , 2013, Nucleic acids research.

[48]  B. Roth,et al.  Protonated state of methotrexate, trimethoprim, and pyrimethamine bound to dihydrofolate reductase. , 1983, Archives of biochemistry and biophysics.