Synthetic riboswitches for the conditional control of gene expression in Streptomyces coelicolor.

We have demonstrated the portability of theophylline-dependent synthetic riboswitches for the conditional control of gene expression in Streptomyces coelicolor. The riboswitches mediate dose-dependent, up to 260-fold activation of reporter gene expression. Riboswitch regulation is a simple method requiring a sequence of only ~85 nt to be inserted between a transcriptional start site and the start codon; no additional auxiliary factors are necessary. The promoters galP2, ermEp1 and SF14 worked well in concert with the riboswitches. They allowed theophylline-dependent expression of not only the heterologous β-glucuronidase reporter gene but also dagA, an endogenous agarase gene. The successful combination of all tested promoters with the riboswitches underlines the orthogonality of riboswitch regulation. We anticipate that any additional natural or synthetic promoters can be combined with the riboswitch.

[1]  J. Gallivan,et al.  A Riboswitch-Based Inducible Gene Expression System for Mycobacteria , 2012, PloS one.

[2]  C. Thompson,et al.  Construction of thiostrepton-inducible, high-copy-number expression vectors for use in Streptomyces spp. , 1995, Gene.

[3]  A. Pardi,et al.  High-resolution molecular discrimination by RNA. , 1994, Science.

[4]  F. Denis,et al.  Adaptation of the Highly Productive T7 Expression System to Streptomyces lividans , 2009, Applied and Environmental Microbiology.

[5]  S. K. Desai,et al.  Synthetic Riboswitches That Induce Gene Expression in Diverse Bacterial Species , 2010, Applied and Environmental Microbiology.

[6]  C. Thompson,et al.  Autogenous transcriptional activation of a thiostrepton‐induced gene in Streptomyces lividans. , 1993, The EMBO journal.

[7]  Mark A. Miller,et al.  Riboswitches for Intracellular Study of Genes Involved in Francisella Pathogenesis , 2012, mBio.

[8]  C. Thompson,et al.  Thiostrepton-induced gene expression in Streptomyces lividans , 1989, Journal of bacteriology.

[9]  M. Bibb,et al.  Cloning and analysis of the promoter region of the erythromycin resistance gene (ermE) of Streptomyces erythraeus. , 1985, Gene.

[10]  R. Pérez-Redondo,et al.  Natural and synthetic tetracycline-inducible promoters for use in the antibiotic-producing bacteria Streptomyces , 2005, Nucleic acids research.

[11]  V. Fedorenko,et al.  β-Glucuronidase as a Sensitive and Versatile Reporter in Actinomycetes , 2011, Applied and Environmental Microbiology.

[12]  Jerry Pelletier,et al.  Inhibition of translation by RNA-small molecule interactions. , 2002, RNA.

[13]  B. Suess,et al.  Streptomyces coelicolor sRNA scr5239 inhibits agarase expression by direct base pairing to the dagA coding region. , 2012, Microbiology.

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

[15]  D. Hodgson,et al.  A Chromosomal Locus Controlling Extracellular Agarase Production by Streptomyces coelicolor A 3(2), and its Inactivation by Chromosomal Integration of Plasmid SCP1 , 1981 .

[16]  J. Fornwald,et al.  Characterization of Streptomyces promoter sequences using the Escherichia coli galactokinase gene. , 1985, Gene.

[17]  Manuela Schuksz Small molecule Interactions with Heparan sulfate , 2009 .

[18]  M. Bibb,et al.  Isolation and characterization of a strong promoter element from the Streptomyces ghanaensis phage I19 using the gentamicin resistance gene (aacC1) of Tn 1696 as reporter. , 1997, Microbiology.

[19]  M. Bibb,et al.  Isolation and characterization of a strong promoter element from the Streptomyces ghanaensis phage I19 using the gentamicin resistance gene (aacC1) of Tn 1696 as reporter. , 1997, Microbiology.

[20]  H. Ikeda,et al.  Hyper-inducible expression system for streptomycetes. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

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

[22]  W R Strohl,et al.  Compilation and analysis of DNA sequences associated with apparent streptomycete promoters. , 1992, Nucleic acids research.

[23]  S. K. Desai,et al.  A high-throughput screen for synthetic riboswitches reveals mechanistic insights into their function. , 2007, Chemistry & biology.

[24]  S. K. Desai,et al.  Genetic screens and selections for small molecules based on a synthetic riboswitch that activates protein translation. , 2004, Journal of the American Chemical Society.

[25]  A. Demain,et al.  Microbial drug discovery: 80 years of progress , 2009, The Journal of Antibiotics.

[26]  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.

[27]  Shana Topp Biological engineering with chemical-sensing macromolecular switches: I. Discovery and applications of small-molecule dependent synthetic riboswitches II. A genetic toolbox for creating reversible Ca2+-sensitive biomaterials , 2009 .

[28]  D. Henner,et al.  Bacillus subtilis requires a "stringent" Shine-Dalgarno region for gene expression. , 1984, DNA.

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

[30]  J. Rabinowitz,et al.  Unique features in the ribosome binding site sequence of the gram-positive Staphylococcus aureus beta-lactamase gene. , 1981, The Journal of biological chemistry.

[31]  F. Schmidt,et al.  Two promoters, one inducible and one constitutive, control transcription of the Streptomyces lividans galactose operon. , 1987, Proceedings of the National Academy of Sciences of the United States of America.

[32]  J. V. Van Impe,et al.  Recombinant protein production and streptomycetes. , 2012, Journal of biotechnology.

[33]  B. Suess,et al.  A theophylline responsive riboswitch based on helix slipping controls gene expression in vivo. , 2004, Nucleic acids research.

[34]  T. Kieser Practical streptomyces genetics , 2000 .

[35]  B. Suess,et al.  Engineered riboswitches: Overview, problems and trends , 2008, RNA biology.

[36]  F. Flett,et al.  High efficiency intergeneric conjugal transfer of plasmid DNA from Escherichia coli to methyl DNA-restricting streptomycetes. , 1997, FEMS microbiology letters.

[37]  Jan van Duin,et al.  Translational initiation on structured messengers : another role for the Shine-Dalgarno interaction , 1994 .

[38]  H. Baylis,et al.  The nucleotide sequence of a 16S rRNA gene from Streptomyces coelicolor A3(2) , 1987, Nucleic acids research.