1 Synthetic Riboswitches that Induce Gene Expression in Diverse 2 Bacterial Species
4 Shana Topp, Colleen M.K. Reynoso, Jessica C. Seeliger, Ian S. Goldlust, Shawn K. Desai,
Dorothée Murat, Aimee Shen, Aaron W. Puri, Arash Komeili, Carolyn R. Bertozzi, June R. Scott, 5 * 6 & Justin P. Gallivan 7
8 MATERIALS AND METHODS
9 Materials. Synthetic oligonucleotides were purchased from Integrated DNA Technologies 10 (Coralville, IA). Culture media was obtained from EMD Bioscience. Theophylline and o-
11 nitrophenyl-β-D-galactopyranoside (ONPG) were purchased from Sigma. Kanamycin was 12 purchased from Fisher Scientific. X-gal was purchased from US Biological. DNA polymerase 13 and restriction enzymes were purchased from New England BioLabs. Plasmid manipulations 14 were performed using E. coli MDS42 cells (Scarab Genomics) that were transformed by
15 electroporation. Purifications of plasmid DNA, PCR products, and enzymatic digestions were 16 performed by using kits from Qiagen. All new plasmids were verified by DNA sequencing 17 performed by MWG Biotech or Elim Biopharmaceuticals. Strains and plasmids are listed in 18 Table S2.
19 Riboswitch development. We first tested whether the most active riboswitch from our 20 previously reported FACS-based screen (14) could be transferred directly to 21 Acinetobacter baylyi. We subcloned this riboswitch into pBAV1K (3) and characterized its 22 performance in A. baylyi and E. coli at various theophylline concentrations. While the switch 23 provides ligand-dependent gene expression in A. baylyi, its dynamic range and activation ratio
24 are diminished (Riboswitch D in Figs. S5 and S6), suggesting that a synthetic riboswitch that has
1 been optimized for small-molecule dependent gene expression in E. coli may not enable optimal
2 function in even a closely related species.
3 To identify synthetic riboswitches that have more desirable properties in A. baylyi, we adapted a
4 previously reported high-throughput screen for use with A. baylyi cells (8, 13). Using this screen,
5 we identified a purine-rich riboswitch sequence that activates gene expression 23-fold for
A. baylyi cells induced with 2 mM theophylline (Fig. S1 and Riboswitch A in Fig. S9). The 6
7 switch also provides 30-fold induction for E. coli cells grown in 2 mM theophylline (Riboswitch
8 A in Fig. S5), suggesting that riboswitches identified in other species may be more portable than 9 those identified in E. coli. To test this idea, we assessed the performance of each riboswitch in 10 the archetypal Gram-positive species, Bacillus subtilis. While the riboswitch identified by FACS
11 in E. coli activates gene expression only 2-fold in B. subtilis (Riboswitch D in Fig. S9), the
12 purine-rich riboswitch identified in A. baylyi activates gene expression greater than 20-fold in 13 B. subtilis (Riboswitch A in Fig. S9).
14 Encouraged by the high portability of the purine-rich riboswitch from our A. baylyi screen,
15 we sought to isolate riboswitches directly in B. subtilis by combining semi-rational design with a
16 low-throughput screen (21). We envisioned that riboswitches identified in this manner would 17 also function in a broader range of bacteria than those identified in E. coli. Guided by data from
18 our previous high-throughput screens in E. coli (13, 14, 22), we designed a 256-member library
19 and isolated Riboswitch B (Fig. S2), which activated gene expression greater than 40-fold in 20 E. coli, A. baylyi, and B. subtilis (Figs. S6, S5, and S9). These data further supported our 21 hypothesis that species other than E. coli may be used to develop highly portable synthetic
1 With this in mind, we wondered if we could introduce rational changes to riboswitches identified 2 in E. coli to enable them to function in other species. Toward this end, we redesigned our
FACS-derived E. coli riboswitch so that it could be ported without further modification to a 3
4 variety of Gram-positive bacteria, which often require stronger binding interactions between the 5 16S rRNA and the Shine-Dalgarno region (Riboswitch E, Fig. S4). Similarly, we applied 6 rational engineering to expand the host cell range of a second riboswitch that we previously 7 identified using an E. coli-based screen (21) (Riboswitch C, Fig. S3). We envisioned that these 8 engineered switches could be combined with Riboswitches A, B, and D (isolated in A. baylyi,
9 B. subtilis, and E. coli, respectively) to create a discrete set of five constructs that could be 10 screened in a novel species to identify the optimal riboswitch for a specific application. 11 E. coli manipulations. Electrocompetent E. coli cells (strain MDS42 (19), Scarab Genomics)
12 were prepared and transformed with the pBAV1K riboswitch constructs by electroporation. All 13 ;-galactosidase assays were performed by the method of Miller (17), using cells that were grown 14 with shaking at 37 ？C, in Luria Broth (LB) containing kanamycin (50 ；g/mL) and 0 or 2 mM
16 A. baylyi manipulations. To transform A. baylyi strain ADP1, ATCC 33305 (10, 23) (gift from
17 Dr. Ichiro Matsumura, Emory University) with the pBAV1K constructs, cells were grown 18 overnight in 5 mL LB at 30 ？C with shaking (250 rpm). In the morning, 3 mL fresh LB was
19 inoculated with 200 ；L from the overnight culture. These cells were grown at 30 ？C for 90 min,
20 at which time the culture was divided into 300 ；L aliquots. 3 ；L plasmid DNA was added to
21 each tube of cells. These cultures were grown with shaking at 30 ？C. After 3 h, 200 ；L of these
22 cultures was plated on selective media. ;-galactosidase assays were performed by the method of
1 Miller (17), using cells that were grown with shaking at 37 ？C, in LB containing kanamycin
2 (50 ；g/mL) and 0 or 2 mM theophylline.
3 B. subtilis manipulations. B. subtilis strain JH642 (2) (gift from Dr. Charles Moran, Emory
4 University) was grown at 37 ?C in LB with shaking (250 rpm). Competent B. subtilis cells were
5 prepared and transformed using the Spizizen method (6). Transformants were selected on LB 6 with 20 ；g/mL kanamycin. To assay for ;-galactosidase activity, strains harboring the
7 riboswitches were grown overnight in LB media with 20 ；g/mL kanamycin. In the morning,
8 cells were diluted 1:100 into fresh selective media containing 1 mM IPTG and either 0 or 2 mM 9 theophylline. ;-galactosidase assays were performed by the method of Miller (17) using 10 permeabilization with toluene.
11 A. baumannii manipulations. Acinetobacter baumannii ATCC 19606 (1) (gift from Dr. Eric
12 Skaar, Vanderbilt University) was grown at 37 ?C in LB with shaking. Electrocompetent cells 13 were prepared by pelleting mid-log phase bacteria at 5000 g for 5 min, washing pellets twice
14 with cold 2 mM CaCl, twice with 10% glycerol, and resuspending the cells in 10% glycerol. 2
15 The electrocompetent A. baumanii cells were transformed using a BioRad electroporator in 0.1 16 cm cuvettes, setting 1.8 kV. The cells were recovered in LB at 37 ?C and selected on LB plates 17 with 30 ；g/mL kanamycin. A. baumannii harboring pBAV1K plasmids were grown at 37 ?C in
18 LB-kanamycin (30 µg/mL) with shaking. All ;-galactosidase assays were performed by the
19 method of Miller (17), using cells that were grown with shaking at 37 ？C, in the presence of 0 or
20 2 mM theophylline.
21 A. tumefaciens manipulations. Competent A. tumefaciens cells (strain C58 (9), gift from Dr.
22 David Lynn, Emory University) were prepared and transformed by the method of Cangelosi et al.
1 (4). Cells were grown in 5 mL LB at 28 ？C with shaking (250 rpm). After 18 h, 10-100 ；L of
2 saturated culture was added to fresh LB (50-150 mL) and allowed to grow until the OD = 0.4. 600
3 The cells were then pelleted three times by centrifugation at 5000 g, washing twice with water
4 and once with 10% glycerol. 1 ；L of plasmid DNA was mixed with 50 ；L of cells, which were
transformed by electroporation at 1800 V (Eppendorf Electroporater 2510; Westbury, NY). 5
6 Electroporated cells were permitted to recover in 500 ；L SOB for 4 h 28 ？C. Finally, 10-100 ；L
of culture was plated on selective media. All ;-galactosidase assays were performed by the 7
8 method of Miller (17), using cells that were grown with shaking at 28 ？C, in the presence of 0 or
9 2 mM theophylline.