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. Author manuscript; available in PMC: 2011 Sep 1.
Published in final edited form as: Nat Struct Mol Biol. 2011 Feb 13;18(3):359–363. doi: 10.1038/nsmb.1989

The glmS Riboswitch Integrates Signals from Activating and Inhibitory Metabolites In Vivo

Peter Y Watson 1, Martha J Fedor 1,*
PMCID: PMC3075592  NIHMSID: NIHMS254763  PMID: 21317896

Abstract

The glmS riboswitch belongs to the family of regulatory RNAs that provide feedback regulation of metabolic genes. It is also a ribozyme that self-cleaves upon binding glucosamine-6-phosphate, the product of the enzyme encoded by glmS. The ligand concentration dependence of intracellular self-cleavage kinetics was measured for the first time in a yeast model system and surprisingly revealed that this riboswitch is subject to inhibition as well as activation by hexose metabolites. Reporter gene experiments in B. subtilis confirmed that this riboswitch integrates positive and negative chemical signals in its natural biological context. Contrary to the conventional view that a riboswitch responds to just a single cognate metabolite, our new model proposes that a single riboswitch integrates information from an array of chemical signals to modulate gene expression according to the overall metabolic state of the cell.

The glmS riboswitch belongs to the family of regulatory RNAs that provide feedback regulation of metabolic genes1–4. Most riboswitches are found in the 5′ untranslated regions of mRNAs that encode metabolic genes, where they control gene expression by adopting alternative RNA structures in the presence and absence of small ligands. Extensive studies of riboswitch interactions with cognate ligands have shown that ligand binding is associated with conformational changes that modulate gene expression by altering the availability of the Shine-Dalgarno sequence, a transcription terminator, or sites associated with RNA processing. In contrast, the glmS riboswitch is a ribozyme that self-cleaves upon binding glucosamine-6-phosphate, the product of the enzyme encoded by glmS in gram-positive bacteria5,6. Riboswitch recognition and activation by cognate metabolite ligands has been studied extensively in vitro, but riboswitch function amidst the complex array of intracellular metabolites with closely related structures has not been analyzed directly. GlcN6P and related compounds participate directly in glmS ribozyme catalysis7 so coenzyme-induced cleavage can be used to monitor metabolite-RNA interactions in vivo.

In order to understand riboswitch function in a biological context, we probed intracellular glmS riboswitch-ligand assembly directly and quantitatively using a system we previously developed for studies of hairpin ribozyme folding in Saccharomyces cerevisiae8,9 (Fig. 1). The ability of exogenous glucosamine to stimulate glmS riboswitch self-cleavage in yeast varied during growth on different carbon sources. The patterns of self-cleavage activity observed during growth in galactose, glucose or glycerol revealed that the glmS riboswitch undergoes inhibition by products of hexose metabolism as well as activation by aminohexose ligands. Expression of a fluorescent reporter gene exhibited the same response to different carbon sources in Bacillus subtilis, the native biological context. Thus, a single ligand-binding site in the glmS riboswitch can integrate information from both activating and inhibitory signals in vivo. The ability of the glmS riboswitch to modulate gene expression in response to the overall metabolic state of the cell might well reflect just a single element of a complex RNA-metabolite interactome.

Figure 1.

Figure 1

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System for measuring intracellular glmS riboswitch cleavage kinetics. (a) The glmS riboswitch from B. anthracis37. Cleavage at the site indicated by the arrow yields 5′ product (5′ P) and 3′ product (3′ P) RNAs. (b) The riboswitch coding sequence was inserted into the 3′ UTR of the yeast PGK1 gene and expressed under the control of a galactose inducible promoter. Full-length glmS chimeric mRNA with a WT riboswitch decays faster than an mRNA with an inactivating G33A mutation10 and is less abundant at steady state due to the contribution of cleavage to decay kinetics9. Comparison of chimeric mRNAs with a WT riboswitch to mRNA with the inactivating G33A mutation ensures that changes in decay kinetics reflect cleavage and not an effect of the sequence insertion on mRNA stability.

Results

Intracellular glucosamine dependence of riboswitch activity

We expressed the glmS riboswitch as a galactose-inducible chimeric mRNA in yeast, which enabled us to determine intracellular cleavage rates from their contribution to intracellular mRNA abundance and turnover kinetics8,9. During yeast growth in galactose, chimeric mRNAs containing the WT glmS riboswitch or a riboswitch with an inactivating G33A mutation10 were expressed at similar levels (Fig. 2, Supplementary Fig. 1), indicating that the intracellular metabolite pool did not support cleavage under these growth conditions. After addition of glucosamine (GlcN), however, full length chimeric mRNA with a WT riboswitch became less abundant and RNA fragments appeared with the size expected for cleavage products (Fig. 2a,b). Exogenous GlcN did not affect the abundance of mutationally inactivated chimeric mRNA (Supplementary Fig. 1b), so exogenous GlcN clearly reduced chimeric glmS mRNA abundance by promoting cleavage and not by changing mRNA synthesis or turnover rates.

Figure 2.

Figure 2

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GlcN dependence of glmS riboswitch cleavage in yeast. (a) RNase protection assays revealed full-length (336 nt) and 5′ P and 3′ P cleavage product RNAs (121 and 215 nt respectively) in yeast expressing WT and G1A chimeric mRNAs during growth in media with galactose and 10 mM GlcN along with RNA fragments upstream and downstream of the riboswitch insert in genomic PGK1 mRNA (93 and 70 nt, respectively). Only full-length mRNA was detected in yeast expressing the mutationally inactivated G33A riboswitch. Act1 mRNA (191 nt) was used for normalization. (b) Lower abundance of full-length chimeric mRNAs with WT and G1A riboswitch insertions relative to chimeric mRNA with an inactivating G33A mutation during growth in galactose and different GlcN concentrations reflects GlcN-dependent cleavage. Bars represent the mean and error bars represent the standard deviation of two or more experiments. (c) Intracellular cleavage rates were calculated from the steady state abundance 9 of chimeric mRNAs with WT or G1A riboswitch inserts and fit to the Michaelis-Menten equation to calculate apparent KdGlcN values. Points represent the mean and error bars represent the standard deviations of rates calculated from the data shown in (b). (d) GlcN dependence of glmS riboswitch cleavage kinetics measured under conditions that approximate a physiological ionic environment in vitro. The plot shows rates measured in a representative experiment and error bars represent the standard error of the fit. Reported kmax and Kd,appGlcN values represent the mean and standard deviation of values obtained from three or more experiments.

The cleavage rates that we calculated from decreases in the abundance of uncut chimeric glmS mRNA9 increased with increasing GlcN, as expected (Fig. 2b,c). However, the kmax value of 0.42 min−1 that we calculated from the fit to the Michaelis-Menten equation was one to two orders of magnitude lower than kmax values of 3 min−1 and 42 min−1 measured in vitro with the GlcN6P coenzyme5,11. Both GlcN and GlcN6P have been implicated as coenzymes in vitro7 but GlcN6P supports 1,000 times higher activity than GlcN in vitro12. GlcN taken up by yeast might activate glmS riboswitch cleavage directly, or it might first undergo phosphorylation to generate GlcN6P before activating the riboswitch. An active site guanine, G1, forms a hydrogen bond with the sugar phosphate of GlcN6P12. Substitution of G1 with adenine abolishes this interaction and a glmS riboswitch with a G1A mutation cleaves at similar rates in GlcN and GlcN6P in vitro12. We examined a chimeric mRNA with a G1A mutant riboswitch to learn how the ability to recognize the sugar phosphate affects intracellular cleavage (Fig. 2). The decrease in rates by a factor of three that we measured for the G1A mutant relative to the WT riboswitches in yeast supplemented with GlcN was much less than the 1,000-fold difference in GlcN6P-induced cleavage activity between WT and G1A riboswitches in vitro. WT and G1A riboswitches also exhibited similar GlcN concentration dependence in yeast, with apparent KdGlcN values in the mM range (Fig. 2c). The similarity in intracellular self-cleavage activity between WT and G1A riboswitches suggests that GlcN directly promotes riboswitch self-cleavage in yeast.

The increase in intracellular cleavage rates with increasing exogenous GlcN concentrations reached a plateau near 100 mM GlcN (Fig. 2c). This plateau is not likely to reflect saturation of import or activation of GlcN catabolism, since intracellular GlcN concentrations continue to increase in yeast as media concentrations of GlcN increase up to 7% (w/v) (400 mM)13. Moreover, the Kd,appGlcN value of 23 mM measured in vivo is not significantly different from the Kd,appGlcN value of 38 mM measured in vitro under conditions designed to approximate the intracellular ionic environment (Fig. 2d). Thus, the apparent Kd values measured for both the WT and G1A riboswitches in vivo appear to reflect their affinity for GlcN.

Dependence of riboswitch activity on carbon metabolism

Many riboswitches must assemble into ligand-bound structures during transcription, since gene regulation often depends on transcription terminator or anti-terminator structures2–4. However, some riboswitches alter splicing efficiency or ribosome binding, which would not require co-transcriptional ligand binding. A possible requirement for co-transcriptional folding of ligand-bound RNA is an important aspect of ligand recognition and riboswitch function that had not yet been studied in vivo. To learn if ligand binding must occur during glmS riboswitch transcription in vivo, GlcN was added after transcription was inhibited by transfer of yeast into media containing glucose instead of galactose (Fig. 3a). Chimeric mRNA with the WT ribozyme and the mutationally inactivated G33A ribozyme decayed at the same rates after GlcN addition, suggesting that GlcN did not activate cleavage after transcription inhibition.

Figure 3.

Figure 3

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Chimeric mRNA cleavage and decay kinetics after transcription inhibition in different carbon sources. (a) WT and G33A chimeric mRNA exhibited similar decay rates after transcription inhibition by transfer of yeast from galactose to glucose with 5 mM GlcN. (b) WT chimeric mRNA exhibited faster decay than chimeric mRNA with a inactivating G33A mutation after transcription inhibition in glycerol, reflecting the contribution of riboswitch cleavage. Cleavage rates were calculated from the difference between the decay rate of full-length chimeric mRNA with a G33A riboswitch insert, which reflects degradation through the endogenous mRNA turnover pathway, and chimeric mRNA with a WT riboswitch insert, which decays through both cleavage and normal degradation pathways 9. Addition of 5 mM GlcN after transcription inhibition accelerated decay of chimeric mRNA with a WT riboswitch even further. Decay kinetics for G33A mutant chimeric were the same under all conditions for which WT decay rates are reported, indicating that mRNA turnover through endogenous mRNA turnover pathways was not affected by riboswitch insertions or changes carbon sources. Reported rates represent the mean and standard deviation of values obtained from two or more replicate experiments.

However, this evidence that GlcN did not induce cleavage of the fully transcribed riboswitch was surprising since glmS riboswitches fold into virtually identical structures in the presence and absence of ligand in vitro14,15, so we considered an alternative explanation. A physiological role for riboswitch inhibition has not been considered previously, but glucose-6-phosphate (Glc6P) is a competitive inhibitor of glmS riboswitch cleavage in vitro7. Growth in glucose increases intracellular concentrations of Glc6P16, raising the intriguing possibility that the absence of detectable glmS riboswitch cleavage activity after transfer of yeast into glucose media results from competitive inhibition of cleavage by glucose metabolites in vivo.

Glc6P is an intermediate in the metabolism of both galactose and glucose17 but glycerol metabolism does not proceed through any hexose phosphate intermediates that are likely to interact with the glmS riboswitch18. In striking contrast to the absence of cleavage in glucose media, riboswitch cleavage products appeared and WT chimeric mRNA decay accelerated relative to decay of the inactive G33A mutant upon GlcN addition when transcription was inhibited by transfer into glycerol rather than glucose (Fig. 3b). Thus, GlcN binding and cleavage did occur in full-length chimeric mRNAs even after transcription inhibition, but only during a glycerol chase. This evidence that GlcN induces glmS riboswitch cleavage activity after transcription inhibition in glycerol suggests that the failure of GlcN to induce cleavage in glucose did not reflect a requirement for co-transcriptional folding of a ligand complex, but rather differences in the metabolic state of the cell during growth in glycerol versus glucose.

Furthermore, cleavage occurred in the absence of exogenous GlcN during mRNA decay in glycerol (Fig. 3b), but not during steady-state growth in media with galactose (Fig. 2) or during decay in glucose (Fig 3a). Thus, differences in riboswitch activity during growth in glycerol relative to growth in galactose or glucose did not reflect differences in uptake of exogenous GlcN and must reflect differences in the intracellular concentrations of endogenous activating and inhibitory metabolites during growth in different carbon sources. These differences in riboswitch activity during growth in glycerol, galactose and glucose provide strong evidence that products of hexose metabolism, such as Glc6P, inhibit cleavage by competing with activating ligands for riboswitch binding. We measured a Ki,app of 10 mM for Glc6P inhibition of GlcN-mediated self-cleavage in vitro in reactions designed to approximate intracellular conditions (Fig. 4a). A similar inhibition constant of 3 mM has been reported for Glc6P inhibition of GlcN6P-activated glmS riboswitch self-cleavage in vitro9. We attempted to measure intracellular concentrations of activating and inhibitory metabolites, but could not resolve hexose and aminohexose isomers. However, the intracellular Glc6P concentration of about 2.3 mM reported for yeast grown in glucose19 is on the same order as the inhibition constants measured in vitro, consistent with an inhibitory role for Glc6P in gene regulation by the glmS riboswitch in vivo. Thus, the glmS riboswitch responds to a complex array of both positive and negative signals that arise from carbon metabolism as yeast grow in different environments.

Figure 4.

Figure 4

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Hexoses inhibit riboswitch cleavage in vitro and in B. subtilis. (a) Cleavage rates were measured in 5mM GlcN and varying concentrations of Glc6P under conditions that approximate a physiological ionic environment in vitro. The plot shows rates measured in a representative experiment and error bars represent the standard error of the fit. The reported Ki,appGlc6P values represent the mean and standard deviation of values obtained from two or more experiments. (b) Riboswitch activity in B. subtilis with a GFP-coding sequence fused to the WT (WTGFP) or inactive (G33AGFP) riboswitch. WTGFP showed a significant (*p<0.001) reduction in fluorescence intensity relative to G33AGFP during growth in minimal media with GlcN, indicating that cleavage decreased GFP mRNA abundance, as expected. Fluorescence intensity increased significantly in bacteria with WTGFP (**p<0.005) in media with glucose and GlcN, but did not change in bacteria with G33AGFP (p>0.3), evidence that glucose or a glucose-derived metabolite prevented riboswitch cleavage from reducing WTGFP mRNA abundance. Bars represent the mean and standard deviation of fluorescence emission intensity obtained from six replicate experiments. Statistical significance was determined using a one-tailed Student's t-test.

We validated the biological significance of this finding using green fluorescent protein (GFP) reporter constructs in which riboswitch cleavage reduces fluorescence in B. subtilis. Fluorescence intensity decreased significantly in bacteria with a WT riboswitch reporter grown in media with GlcN relative to reporter mRNA with an inactivating G33A mutation (Fig. 4b), as expected if GlcN-activated self-cleavage reduced reporter mRNA abundance. Furthermore, B. subtilis exhibited enhanced fluorescence during growth in media with both GlcN and glucose, consistent with inhibition of glmS riboswitch self-cleavage by glucose metabolites. The difference in fluorescence did not reflect metabolic conversion of glucose to GlcN because we used a glmS− strain20. Furthermore, no increase in fluorescence occurred in bacteria expressing a reporter with a mutationally inactivated G33A riboswitch, so it did not reflect changes in mRNA synthesis or turnover. Thus, the glmS riboswitch was subject to inhibition by glucose-derived metabolites in gram-positive bacteria, its natural biological context. These results provide the first evidence that metabolites play both stimulatory and inhibitory roles in riboswitch-mediated gene regulation in vivo.

Discussion

Large, unexpected changes in self-cleavage kinetics under different growth conditions revealed inhibition as well as activation by hexose metabolites. Reporter gene experiments in B. subtilis confirmed that the glmS riboswitch integrates positive and negative chemical signals in its natural biological context. These findings demonstrate that glmS riboswitch activity depends not only on the concentration of cognate ligands that promote self-cleavage but also on products of hexose metabolism that inhibit self-cleavage. The ability of intracellular metabolites to block the interaction between a riboswitch and its cognate ligand points to an unanticipated role for riboswitches in integration of multiple chemical signals. Negative feedback regulation of GlmS through riboswitch activation by intracellular GlcN6P is well known5,6. However, our findings provide the first evidence that intracellular products of hexose metabolism block activation by cognate ligands, thereby enabling the glmS riboswitch to modulate gene expression in response to the overall metabolic state of the cell. A previous report that growth of B. subtilis in GlcN reduces GlmS activity, but growth in glucose and GlcN does not21, supports the idea that the riboswitch integrates information from an array of metabolites, not just its cognate ligand. NagB deaminates GlcN6P to generate fructose-6-phosphate, the reverse of the reaction catalyzed by GlmS. NagB is downregulated in glucose and upregulated in GlcN21,22. Intriguingly, our results indicate that the glmS riboswitch plays a role in reciprocal regulation of GlmS, responding to intracellular hexose and aminohexose metabolites to achieve both feed-forward upregulation and feedback downregulation of GlmS expression (Fig. 5).

Figure 5.

Figure 5

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Model for riboswitch regulation of GlmS gene expression. Riboswitch cleavage activity integrates information about the metabolic state of the cell by responding to the concentrations and affinities of an array of chemical signals. In this model, hexoses increase glmS mRNA abundance and upregulate GlmS expression by inhibiting riboswitch cleavage (green line), while aminohexoses decrease glmS mRNA abundance and downregulate GlmS expression by activating riboswitch cleavage (red line). The dashed box illustrates the former model that a single cognate ligand, GlcN6P, activates the riboswitch to down-regulate GlmS expression.

These findings have broad implications for mechanisms of riboswitch-mediated gene regulation. Tandem riboswitch arrangements have been shown to respond to multiple metabolites23 but this is the first evidence that a single riboswitch responds to multiple chemical signals, both activating and inhibitory. The ability of a single riboswitch to integrate information from multiple chemical signals may well extend to other riboswitches. The SAM-I riboswitch, which regulates ratios of S-Adenosylmethionine (SAM) to S-adenosyl-L-homocysteine (SAH)1,24, provides one illustration. The P1 and P3 helices of the SAM-I riboswitch interact with different functional groups of SAM to stabilize a specific tertiary structure and promote folding of a transcription terminator. SAH forms the same interaction with the P3 helix as SAM but lacks a key interaction with the P1 helix24,25. If the SAM riboswitch integrates positive and negative chemical signals like the glmS riboswitch, abundant SAH might compete with SAM for P3 binding without stabilizing the interhelical tertiary structure or promoting folding of the transcription terminator. Indeed, SAM-I riboswitch variants with similar affinities for SAM in vitro have different effects on downstream genes in vivo, which might reflect different susceptibilities to inhibition by SAH or related metabolites in vivo26. Thus, the SAM-I riboswitch might integrate information about SAM and SAH abundance in the same way that the glmS riboswitch integrates information from carbon metabolism.

We found that the glmS riboswitch responds to both inhibitory and activating metabolites in yeast and bacteria. Our finding that glmS riboswitch activity is modulated by several metabolites in vivo, not just a single cognate ligand, suggests an expanded physiological role for regulatory RNAs in integrating information from an array of chemical signals. We propose a new paradigm for riboswitch-mediated gene regulation in which ligand binding reflects the concentrations and affinities of an array of chemical signals and serves to integrate information about the metabolic state of the cell. The glmS riboswitch response to multiple metabolites that we describe here likely represents an early indication of a vast RNA-metabolite interactome that remains to be explored.

Methods

Plasmid construction and propagation

Oligonucleotide primer sequences are shown in Supplementary Table 1. A plasmid template for transcription of the glmS ribozyme, pTS, was created by fusing the ribozyme sequence from B. anthracis to a T7 RNA polymerase promoter, introducing flanking BamH I and EcoR I sites using PCR, and insertion into pUC1827. The glmS ribozyme sequence was inserted into the 3′ UTR of the yeast PGK1 gene in pGS, a derivative of pRS31628, for expression of chimeric glmS PGK1 mRNAs in yeast. pGS was prepared from pAMGAL28, a plasmid previously used for galactose-inducible expression of chimeric hairpin ribozyme mRNAs in yeast8,28,29. To construct pGS, an Mlu I site was inserted upstream of the ribozyme sequence in pTS using QuikChange™ mutagenesis (Stratagene) and an Afl II-Mlu I fragment encoding the glmS ribozyme was inserted into the 3′ UTR of the PGK1 gene.

The plasmid template for transcription of hybridization probes, pGAp, contained sequences corresponding to an Nhe I and Bgl II fragment of yeast ACT1 mRNA and chimeric glmS PGK1 mRNA. pGAp was derived from pGEM4-Z using conventional procedures as described previously28.

pBGSEF, a derivative of pAD43-2530 (Bacillus Genetic Stock Center) that contains the complete glmS 5′ UTR fused to a GFP coding sequence was constructed to express GFP under glmS ribozyme control in B. subtilis. A fragment containing the complete 5′ UTR and the glmS ribozyme coding sequence from B. anthracis was inserted into pAD43-25 using QuikChange™ mutagenesis31. G1A and G33A mutations were introduced into pTS, pGAp, pGS, and pBGSEF using QuikChange™ mutagenesis. Plasmids were propagated in E. coli strains XL1-Blue or XL10-Gold (Stratagene), S. cerevisiae strain HFY114 (MATa ade2-1 his3-11,15 leu2-3 112 trp1-1 ura3-1 can1-100)32, or B. subtilis strain 60984 (glmS2 metC7 trpC2) 20 (Bacillus Genetic Stock Center)

Intracellular self-cleavage kinetics

Intracellular cleavage assays were performed as previously described9, except that minimal galactose media was maintained at pH 7 using phosphate buffer (50mM PO4). Glucosamine (pH 7.0, 30°C) was added at the indicated concentrations, and cultures were incubated for 1 hr at 30 °C before extracting RNA. Intracellular cleavage rates can be calculated from the relative abundance of WT and mutationally inactivated chimeric mRNAs at steady state8,28,29. Reported values represent the mean, and errors bars represent the standard deviation, of two or more experiments. Cleavage rates were fit to the Michaelis-Menten equation, and reported kinetic parameters represent the constants and errors of the fit.

Time course experiments for measuring chimeric mRNA decay kinetics were initiated by transferring yeast cultures from minimal media with 2% (w/v) galactose to minimal media with either 2% (w/v) glucose or 3% (v/v) glycerol. Chimeric mRNAs were quantified using RNase protection assays and normalized with respect to yeast ACT1 mRNA, as described9. Intracellular chimeric mRNA decay rates were calculated from the fit to a single exponential rate equation. Intracellular cleavage rates were calculated from the difference between decay rates of chimeric mRNAs with a WT riboswitch insert or a riboswitch insert with an inactivating G33A mutation. The plots shown in the figures represent typical decay time course experiments. Reported values represent the mean and standard deviation of two or more experiments.

In vitro cleavage kinetics

32P-labeled WT and G1A riboswitch RNAs were transcribed from linearized pTS and pTSG1A plasmids using T7 RNA polymerase and [α32P ATP] as described33, except that reactions contained 20 mM NaHEPES pH 7.8 instead of TrisCl, which promotes glmS riboswitch cleavage7. Transcripts were fractionated by denaturing gel electrophoresis and eluted. Full-length riboswitch RNAs were converted into sodium salts by DEAE-650M chromatography (Toyopearl), precipitated with ethanol and dissolved in 50 mM NaHEPES pH 7.5.

To measure kinetic parameters for GlcN-activated self-cleavage, RNA solutions were heated to 85 °C for 1 min., cooled to 30 °C, and brought to final concentrations of 50 mM NaHEPES pH 7.5, 2 mM MgCl2, 150 mM NaCl, and 0.1 mM EDTA. GlcN (pH 7.5) was added to reach the indicated concentrations. Aliquots were removed at intervals over one hour, quenched by the addition of 90% (v/v) formamide, 25 mM EDTA, 0.002% (w/v) xylene cyanole and 0.002% (w/v) bromophenol blue, and fractionated by denaturing gel electrophoresis. Cleavage rates were calculated from the fit to a single exponential rate equation34. Kd,appGlcN values were calculated from the fit to Kd,appGlcN = kmax × [GlcN])/([GlcN] + Kd,app. Reported values represent the mean and standard deviation from three experiments.

Inhibition constants were determined from cleavage rates measured in reactions with 5 mM GlcN and the indicated concentrations of Glc6P. The IC50Glc6P was calculated from the fit to a sigmoid curve: kcleavage=kmin + (kmax)/(1+e((Ki−[Glc6P])*C)), where C is the slope of the Hill plot of [Glc6P] versus kobs,cleavage. The Ki,app was calculated using the Cheng-Prusoff equation35 and the Kd,appGlcN value measured under the same conditions.

GFP fluorescence in B. subtilis

B. subtilis was transformed36 with plasmids encoding WTGFP or G33AGFP riboswitch-GFP fusion proteins. We used B. subtilis strain 60984, which has a glmS2 mutation that prevents synthesis of GlcN6P from glucose to facilitate manipulation of intracellular hexose and aminohexose metabolite concentrations. Cells were grown overnight with agitation at 37 °C in minimal media supplemented with 0.5% (w/v) GlcN, pelleted by centrifugation then resuspended in minimal media with 0.5% (w/v) glucose or 0.5% (w/v) GlcN as indicated, to an absorbance at 600 nm (A600) of 0.05 or lower. Cells were grown at 37 °C to mid log phase (A600 ∼ 0.3), pelleted, resuspended in fresh media containing the same concentrations of glucose and GlcN to obtain an A600 = 0.15, and grown again to mid-log phase (A600 ∼ 0.3). Samples (20 μL) were transferred into a 384-well black microplate (Greiner), excited at 460 nm, and emission intensity was measured at 511 nm in a SpectraMax M2e fluorescent plate reader (Molecular Devices). Emission intensity was normalized to the absorbance at 600 nm of each sample, and background emission from untransformed cells was subtracted. Reported data represent the mean and standard deviation of fluorescence emission intensity obtained from six replicate experiments.

Supplementary Material

1

Acknowledgments

This work was supported by NIH Grant RO1 GM062277. P.Y. Watson was supported by a graduate fellowship from The Skaggs Institute for Chemical Biology. We thank Joseph Pogliano for advice regarding the B. subtilis experiments, Julia Viladoms and Manami Saha for assistance with plasmid constructions, and JV for critical reading of the manuscript.

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