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. Author manuscript; available in PMC: 2018 Jan 19.
Published in final edited form as: Mol Cell. 2016 Dec 15;65(2):220–230. doi: 10.1016/j.molcel.2016.11.019

Metabolism of Free Guanidine in Bacteria is Regulated by a Widespread Riboswitch Class

James W Nelson 1,6, Ruben M Atilho 2,6, Madeline E Sherlock 2,6, Randy B Stockbridge 3,4, Ronald R Breaker 1,2,5,*
PMCID: PMC5360189  NIHMSID: NIHMS849042  PMID: 27989440

SUMMARY

The guanidyl moiety is a component of fundamental metabolites including the amino acid arginine, the energy carrier creatine, and the nucleobase guanine. Curiously, reports regarding the importance of free guanidine in biology are sparse and no biological receptors that specifically recognize this compound have been previously identified. We report that many members of the ykkC motif RNA, the longest-unresolved riboswitch candidate, naturally sense and respond to guanidine. This RNA is found throughout much of the bacterial domain of life, where it commonly controls the expression of proteins annotated as urea carboxylases and multidrug efflux pumps. Our analyses reveal that these proteins likely function as guanidine carboxylases and guanidine transporters, respectively. Furthermore, we demonstrate that bacteria are capable of endogenously producing guanidine. These and related findings demonstrate that free guanidine is a biologically relevant compound and several gene families exists that can alleviate guanidine toxicity.

INTRODUCTION

Guanidine was first discovered as a thermal decomposition product of the nucleobase guanine (Strecker, 1861). Industrial-scale production of guanidine has been exploited for the manufacture of explosives and other chemicals, but this compound is perhaps best known to the scientific community as a commonly-used reagent for protein denaturation (Greene and Pace, 1974). Microbes capable of metabolizing guanidine have been previously studied, in part due to the interest in guanidine bioremediation (Bierema, 1909; Ebisuno and Takimoto, 1981; Iwanoff and Awetissowa, 1931; Kihara et al., 1955; Mitchell, 1985, 1987). Guanidine also has been proposed to be generated by human cells as part of the guanidine cycle via the cleavage of canavanine (Kihara et al., 1955; Natelson and Sherwin, 1979). Despite these scattered findings, guanidine has never been considered to be a major cellular metabolite. Indeed, no biological receptor has previously been identified that specifically recognizes guanidine.

In the current study, we determined that many bacteria employ riboswitches (Breaker, 2011; Serganov and Nudler, 2013) to selectively bind guanidine (most likely as the guanidinium cation) and regulate genes encoding proteins that overcome its toxicity. Over the past fifteen years, more than 30 different riboswitch classes have been demonstrated to control a variety of metabolic or physiological processes, including previously underappreciated aspects of biology. Typically, widespread bacterial riboswitches recognize metabolites or signaling molecules that are themselves widespread and broadly important to life.

One of the most common uncharacterized RNA motifs identified in bacteria over the past two decades is the ykkC riboswitch candidate. This RNA motif was one of eight classes discovered in an earlier study using comparative sequence analysis to identify riboswitches (Barrick et al., 2004). Seven of the motifs identified in this previous study have since been characterized, and six of these were found to control important aspects of biology related to cell wall biosynthesis (Winkler et al., 2004; Nelson et al., 2013) and sporulation (Marchais et al., 2011; Nelson et al., 2013), Mg2+ homeostasis (Dann et al., 2007), Mn2+ homeostasis (Dambach et al., 2015; Price et al., 2015), glycine metabolism (Mandal et al., 2004), and biosynthesis of modified nucleotides (Roth et al., 2007). Some of these RNA candidates were considered “orphan” riboswitches because their ligands had not immediately been validated experimentally (Block et al., 2010; Meyer et al., 2011). The ykkC motif RNA is the last orphan remaining from this initial collection of RNA classes (Barrick et al., 2004) to resist all previous efforts at experimental validation (Meyer et al., 2011).

Members of the ykkC class control an eclectic distribution of genes including those encoding predicted urea carboxylases and their associated proteins, as well as predicted allophanate hydrolases, arginases, creatininases, and nitrate/sulfate/bicarbonate transporters (Barrick et al., 2004; Meyer et al., 2011). The RNA is also commonly found upstream of genes annotated as emrE or sugE, which are predicted to encode small multidrug resistance (SMR) efflux pumps (Jack et al., 2000; Schuldiner, 2009). For example, the Escherichia coli EmrE protein has been demonstrated to be highly indiscriminate and is thought to have evolved to protect the cell against a wide variety of small molecule toxins (Schuldiner, 2012). In addition, some ykkC motif RNAs are found upstream of genes involved in branched-chain amino acid biosynthesis and purine metabolism (Meyer et al., 2011). Frequently, these RNAs are predicted to control the entire de novo purine biosynthetic operon of their host organism. Due to the diversity of genes associated with this motif, no single compound emerged as a probable riboswitch ligand.

We hypothesized that ykkC RNAs might recognize some toxic ligand, as they are associated with disparate metabolic genes and genes that encode numerous types of transporter proteins. Accordingly, we screened a wide variety of growth conditions, including exposure to numerous toxic compounds, for those that trigger riboswitch activation in cells. Of the roughly 2,000 conditions tested, only the addition of guanidine hydrochloride led to riboswitch-mediated gene expression. We subsequently demonstrated that certain ykkC motif RNAs are capable of specifically recognizing guanidine in vitro at concentrations many orders of magnitude below those required for guanidine to non-specifically affect RNA structure. Furthermore, biochemical analysis of a previously studied urea carboxylase (Kanamori et al., 2004) revealed that this protein prefers guanidine over urea as a substrate. Additionally, examination of one of the SugE transporters whose expression is controlled by the riboswitch provided evidence that it selectively recognizes guanidine. Finally, we demonstrated that bacteria are capable of endogenously producing guanidine under laboratory conditions. These and other findings reveal that the majority of ykkC motif RNAs are members of a broadly distributed riboswitch class that specifically senses guanidine and controls the expression of a variety of genes that together constitute a previously unknown super-regulon for its metabolism and transport.

RESULTS

The ykkC Motif is a Widespread Orphan Riboswitch Candidate Predicted to Control the Expression of a Variety of Transporter and Metabolic Genes

By performing a series of homology searches, we identified ~2000 distinct ykkC RNA sequences in Actinobacteria, Firmicutes, Proteobacteria and Cyanobacteria (Dataset S1). Through additional experimental efforts as discussed below, we established that there are at least two distinct subtypes of ykkC motif RNAs. Most of these RNAs are of the predominant “subtype 1” (Figure 1A), whereas a rarer variant ykkC motif called “subtype 2” (Figure S1) carries prevalent alterations in three otherwise highly-conserved regions of the ykkC motif (Figure S1A). Analysis of the nucleotide sequences and structures located 3′ of this motif revealed the frequent presence of an adjoining expression platform. Therefore, we predicted that the vast majority of ykkC motif representatives of both subtypes function as riboswitches by activating expression of downstream genes upon recognition of the cognate ligand by, for instance, precluding formation of an intrinsic transcription terminator stem (Gusarov and Nudler 1999; Yarnell and Roberts, 1999).

Figure 1. The ykkC Motif is Widespread in Bacteria.

Figure 1

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(A) Consensus sequence and secondary structure model derived from ~1500 subtype 1 ykkC motif RNAs. Asterisks identify nucleotides that differ between subtypes 1 and 2.

(B) Genes predicted to be controlled by subtype 1 ykkC motif RNAs.

See also Figure S1.

The identities of the ligands recognized by newly discovered riboswitch classes frequently can be inferred based on the functions of proteins whose genes reside immediately downstream. Unfortunately, the total collection of genes associated with ykkC motif RNAs (Figure 1B, Figure S1B) did not suggest any likely ligand candidates in addition to those that already had been examined (Meyer et al., 2011; Figure S2 legend). Intriguingly, two other bacterial RNA motifs, called mini-ykkC (Weinberg et al., 2007) and ykkC-III (Weinberg et al., 2010), are often found upstream of genes similar to those associated with subtype 1 RNAs. This suggests that these three RNA motifs might all respond to the same ligand, either directly or by employing an intermediary protein factor. We therefore anticipated that any insight gained by the study of subtype 1 RNAs would also advance our understanding of the biological and biochemical functions of these other two RNA classes as further discussed below.

Addition of Guanidine Triggers Gene Expression Mediated by a Subtype 1 ykkC Motif RNA

In the current study, we screened a larger diversity of compounds and growth conditions for any factor that might result in activation of a riboswitch reporter construct. We used a reporter created previously (Meyer et al., 2011) by fusing the subtype 1 ykkC motif representative naturally associated with the Bacillus subtilis ykkC coding region to a β-galactosidase (lacZ) reporter gene (Figure 2A). Of the nearly 2,000 conditions examined, only the addition of guanidine triggered reporter gene expression mediated by the ykkC motif RNA (Figure S2A).

Figure 2. A ykkC Motif RNA Responds to Guanidine and Controls the Expression of a Transporter that Alleviates Guanidine Toxicity.

Figure 2

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(A) Sequence and predicted secondary structure of the ykkC-reporter construct based on the ykkC motif representative associated with the B. subtilis ykkCD operon. Red nucleotides are >97% conserved. The secondary structure depicted is proposed to form in the absence of the riboswitch ligand. As with many ykkC motif RNAs, part of the predicted aptamer region overlaps a downstream terminator stem such that the presence of the riboswitch ligand will disrupt terminator stem formation, thereby resulting in increased reporter expression. Mutations of conserved nucleotides and secondary structures are designated M1 through M3.

(B) Plot of β-galactosidase reporter gene expression in B. subtilis versus the concentration of guanidine, wherein expression in the absence of added guanidine is set to 1. Data points are the average of three measurements and are representative of experiments performed on multiple days. Error bars indicate the standard deviation of the measurements, and when not present are smaller than the data point. Structure for guanidinium is depicted, as this is the predominant form of guanidine under the assay conditions.

(C) Reporter gene expression of B. subtilis containing wild-type (WT) and mutant reporter constructs as described in A when grown on Lysogeny Broth (LB) agar containing 10 mM guanidine and 100 μg mL−1 X-gal.

(D) Agar diffusion assay of wild-type and ykkCD knockout (ΔykkCD) B. subtilis cells grown on the same LB agar plate. Paper disk (center) was spotted with 10 μL of a 6 M guanidine hydrochloride solution (pH 4.7 at 23°C).

(E) Plot of the β-galactosidase expression levels for WT (set to 1 when measured in the lowest guanidine concentration) and ΔykkCD B. subtilis cells at various concentrations of guanidine. Data are the average of three measurements and are representative of experiments performed on multiple days. Error bars represent the standard deviation of the measurement. Other annotations are as described in B.

(F) Agar diffusion assays of B. subtilis WT and ΔykkCD cells. Disks were spotted with 10 μL of a 6 M guanidine solution. Both plates contained LB medium supplemented with 100 μg mL−1 X-gal. Notably for ΔykkCD cells, we observe an inner halo of growing bacteria that do not exhibit reporter gene expression in the presence of high guanidine levels. We have observed a similar phenomenon for other bacteria bearing riboswitch reporters in the presence of toxic compounds (Kim et al., 2015).

See also Figures S2 and S3.

To experimentally confirm this hit, we grew the B. subtilis reporter strain with various concentrations of added guanidine (Figure S2B). Reporter gene expression progressively increases with increasing guanidine, beginning with concentrations as low as 100 μM. Since guanidine is an effective protein denaturant above 1 M (Greene and Pace, 1974), this suggests that guanidine’s influence on reporter gene expression is unlikely to be due to nonspecific denaturation of a riboswitch or protein regulatory factor.

Guanidine-dependent expression also occurs if the native promoter for the ykkC gene (Meyer et al., 2011) is replaced with an unrelated promoter (Figure 2B), which indicates that the ykkC motif RNA is responsible for ligand-induced gene regulation. Furthermore, disruption of conserved RNA sequence or structural features, such as in reporter constructs M1 and M2, results in a loss of expression (Figure 2C). In contrast, incorporation of mutations in construct M3 that retain a conserved RNA structure also permits guanidine-dependent regulation.

To further demonstrate that guanidine selectively triggers RNA-mediated gene expression, we created another reporter construct in E. coli cells based on a representative subtype 1 example associated with the Klebsiella pneumoniae tauA gene, which encodes a putative ATPase located near additional genes commonly associated with ykkC motif RNAs. Again, the addition of guanidine to the culture medium activates expression of the WT reporter construct, whereas mutation of a single conserved nucleotide in construct M4 disrupts regulation (Figure S3A–C). Furthermore, guanidine does not trigger expression of a member of an unrelated riboswitch class (Figure S3D–E). These results are consistent with the hypothesis that subtype 1 representatives of ykkC motif RNAs function as guanidine-dependent riboswitches, and hereafter these RNAs will be called guanidine class I or ‘guanidine-I’ riboswitches.

The guanidine-I riboswitch representative examined from B. subtilis controls the expression of an apparent two-gene operon comprised of the ykkC and ykkD coding regions. The proteins encoded by these genes are predicted to form a heterodimer and function as an SMR efflux pump (Jack et al., 2000). If guanidine is the biologically-relevant ligand for this riboswitch class, then this protein complex might function to alleviate guanidine toxicity by reducing its concentration in cells. Indeed, upon deleting the ykkC and ykkD genes, we observed decreases in both the concentrations of guanidine required to inhibit bacterial growth (Figure 2D) and to activate riboswitch-mediated expression of lacZ (Figures 2E and 2F).

As noted earlier, guanidine is a well-known chaotropic agent, and therefore might adversely affect RNA structure in a manner similar to its protein denaturation mechanism. Alternatively, structural reorganization by ionic stabilization of the RNA could be mediated by guanidinium cations (Figure 2B), which represent the predominant form of guanidine at biologically-relevant pH conditions. Metal and nonmetal (e.g. amine and polyamine) cations are well known to interact non-specifically with RNA and induce structural changes (Hermann and Westhof, 1999; Pyle, 2002). Given these potential modes of non-specific interaction, we sought to provide several additional lines of evidence that guanidine is the biologically relevant ligand as described in the following sections.

Molecular Recognition Determinants for Guanidine Binding by a Riboswitch

The determinants of guanidine recognition were examined by testing a number of chemically related compounds both for reporter gene regulation and for binding in vitro. Of the 16 guanidine analogs examined using the B. subtilis riboswitch reporter (Figure 2A), none resulted in expression equal to that induced by guanidine (Figure S3F). Only the closest chemical analogs of guanidine, specifically hydroxy-, amino-, and methylguanidine, yielded reporter gene expression above background. Of these, hydroxyguanidine is unstable, and yields guanidine upon degradation (Walker and Walker, 1959). Therefore, it is possible that the signal generated by this compound is not caused by the original analog. Strikingly, the addition of up to molar amounts of urea failed to yield any measurable increase in reporter gene expression. These results suggest that the ykkC motif RNAs examined above have evolved to serve as highly selective sensors for free guanidine, and to exclude even closely-related analogs such as urea that might otherwise inappropriately trigger gene expression. It seems unlikely that the riboswitch might preferentially respond to another natural compound that incorporates a guanidyl moiety since any modification to a nitrogen atom causes a loss of affinity.

We considered the possibility that guanidine might induce cells to produce a structurally unrelated signaling compound, or perhaps a metabolic byproduct of guanidine degradation, that then directly triggers riboswitch function. To determine whether the riboswitch aptamer directly recognizes guanidine, we conducted in-line probing assays (Regulski and Breaker, 2008; Soukup and Breaker, 1999) to monitor RNA structural changes that are indicative of riboswitch modulation by ligand binding. Initially, we examined a 104 nucleotide aptamer called 104 uca (Figure 3A) that was derived from the guanidine-I riboswitch associated with the putative urea carboxylase gene from the bacterium Nitrosomonas europaea. Guanidine causes decreased levels of spontaneous RNA strand scission at two sites (Figure 3B), indicating that specific nucleotides within these conserved regions undergo structural reorganization. A dissociation constant (KD) of ~60 μM for the guanidine-RNA complex is estimated from these data (Figure 3C). These results are typical of the structural changes that occur when representatives of other riboswitches classes are exposed to their cognate ligands. In contrast, 20 different compounds carrying a guanidyl moiety or a charged amine group were also examined by in-line probing (Figure S4), but only guanidine triggers changes in the shape of the RNA aptamer.

Figure 3. Ligand Binding by a Guanidine-I Riboswitch Aptamer.

Figure 3

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(A) Sequence and secondary structure of the 104 uca RNA construct derived from the uca gene of N. europaea. Lowercase letters are nucleotides added to promote efficient in vitro transcription with T7 RNA polymerase. Data from the in-line probing assay depicted in B were used to map regions of constant and reduced RNA scission. Arrowhead denotes the start of mapping of available data on the gel.

(B) Polyacrylamide gel electrophoresis (PAGE) analysis of the products of in-line probing of 5′ 32P-labeled 104 uca RNA in the presence of added guanidine hydrochloride. NR, T1, and −OH, respectively, designate RNA undergoing no reaction, or RNAs that have been partially digested with ribonuclease T1 (cleaves after each G), or partially digested under alkaline conditions (cleaves after every nucleotide). Bands corresponding to cleavage after certain G residues in the sequence are annotated. Guanidine concentrations were progressively increased from no ligand added (−) to 10 mM (far right lane). Regions 1 and 2 undergo guanidine-dependent structure stabilization.

(C) Plot of the fraction of RNA bound by guanidine as inferred from the normalized fraction of spontaneous RNA scission at regions 1 and 2 (as depicted in A and B) relative to the logarithm of the molar concentration of guanidine. The solid line is an idealized binding curve expected for a 1-to-1 interaction between a receptor and its ligand. Data shown are representative of multiple experiments.

See also Figure S4 through S7.

The structural changes observed with the 104 uca RNA are unlikely to be due to the chaotropic nature of guanidine. Spontaneous RNA strand scission is reduced by guanidine, which indicates that the RNA is becoming more rather than less structured. The latter effect would be expected if guanidine were functioning as a denaturant. Also, the KD for RNA binding is more than five orders of magnitude lower than is necessary to cause protein denaturation (Greene and Pace, 1974). Moreover, ligand-mediated modulation occurs with a dose-response curve consistent with a 1-to-1 binding interaction (Figure 3C). This suggests that the mechanism of riboswitch function is due to the docking of a single ligand in a single saturable binding site, rather than by non-specific neutralization of negatively charged phosphate groups on the RNA backbone by guanidinium.

For other riboswitch classes, many highly-conserved nucleotides within the aptamer domain establish critical structural contacts to form a selective ligand binding pocket. Therefore, if guanidine is being selectively bound by the RNA, then the mutation of any single conserved nucleotide is likely to adversely affect ligand-induced riboswitch function. To test this hypothesis, we examined a larger collection of mutant 104 uca aptamers (Figure S5, constructs M8 through M16) that individually alter nine highly-conserved nucleotides. In each instance, the single mutation either reduces (M10 and M12) or eliminates (M8, M9, M11, M13–M16) guanidine binding. These results suggest that guanidine-I riboswitches exploit numerous conserved nucleotides to form a selective binding pocket for guanidine. Indeed, x-ray structural analysis of a guanidine-I riboswitch aptamer confirms the critical roles of all highly-conserved nucleotides to form a 1-to-1 complex between RNA and guanidine (Reiss and Strobel, 2016).

Similar ligand-induced structural modulation of other guanidine-I riboswitch aptamers also is observed (Figures S6 and S7). In contrast, the addition of up to 1 mM guanidine to a subtype 2 ykkC motif RNA (data not shown) or 0.5 M guanidine to aptamers from four other riboswitch classes (azaaromatic, c-di-AMP, c-AMP-GMP, ZTP; data not shown) failed to induce structural changes. These results again indicate that guanidine, even at exceedingly high concentrations, does not cause RNA structural changes by non-specific interactions. Importantly, the addition of guanidine to representative mini-ykkC and ykkC-III motif RNAs also induces structure modulation (Breaker Laboratory, data not shown). These preliminary findings are consistent with our original hypothesis that mini-ykkC and ykkC-III motif RNAs might represent additional distinct riboswitch classes that respond to the same ligand as ykkC motif RNAs with similar gene associations (Weinberg et al., 2010).

Guanidine Selectively Induces Riboswitch-mediated Transcription

The ability of guanidine to selectively regulate transcription in vitro was assessed by measuring the extent of transcript elongation of several guanidine-I riboswitch constructs in the presence of guanidine or various analogs. Each construct has the potential to form a strong intrinsic terminator stem, but ligand binding presumably results in a stable aptamer structure that precludes formation of the terminator. In single-round transcription reactions with a riboswitch construct derived from the region upstream of the smr gene from the bacterium Desulfotomaculum ruminis (Figure 4A), the inclusion of guanidine ranging from 1 to 100 mM yields progressively greater amounts of full-length RNA transcript (Figure 4B). Similar results are observed for the guanidine-I riboswitches associated with the Clostridium difficile emrE (Figure S8A) and B. subtilis ykkC (Figure S8B) genes. Mutant construct M5, which carries a nucleotide change at a highly conserved position, exhibits a complete loss of guanidine responsiveness (Figure 4C). Likewise, mutations M6 and M7 predictably diminish and restore, respectively, the guanidine-responsive transcription of the smr construct.

Figure 4. Guanidine Regulates Riboswitch-dependent Transcript Termination.

Figure 4

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(A) Sequence and secondary structure model of the guanidine-I riboswitch construct derived from the smr gene of D. ruminis and used for in vitro transcription assays. Annotations are as described in the legend for Figure 2A. Terminated transcripts end within the U-rich tract immediately following the terminator stem, whereas full-length transcripts include the 63 nucleotides following the U-rich tract.

(B) Plot of the fraction of full-length (FL) smr riboswitch transcripts contributing to the total number transcripts [FL plus terminated (T)] generated by single-round transcription reactions versus the logarithm of the molar concentration of guanidine. The curve is an idealized representation of the results expected for a 1-to-1 interaction between guanidine and the riboswitch given the observed minimum and maximum values for fraction elongated. Inset: PAGE analysis used to generate the data plotted. Data shown here and in parts C and D are representative of repeated experiments performed on different days.

(C) (Top) PAGE analysis of single-round transcription termination assays of WT and various mutant smr riboswitches. (Bottom) Plot of the fraction of FL RNA generated as derived from the PAGE data.

(D) (Top) PAGE analysis of single-round transcription termination assays of WT smr riboswitches in the absence of ligand (−), in the presence of guanidine or its analogs (10 mM), or in the presence of urea (0.5 M). (Bottom) Plot of the fraction of FL RNA generated as derived from the PAGE data.

See also Figure S8.

Notably, guanidine has no effect on the extent of transcript elongation for a riboswitch that recognizes a different ligand (Figure S8C; Kim et al., 2015). Finally, the addition of structural analogs of guanidine, including urea, does not lead to transcript elongation (Figure 4D). These data demonstrate that the riboswitches tested are capable of specifically recognizing guanidine in vitro and that ligand binding by the RNA is sufficient to control the expression of downstream genes.

A Urea Carboxylase Enzyme Prefers Guanidine Over Urea

We speculated that the genes controlled by guanidine-I riboswitches represent a super-regulon that is capable of reducing guanidine toxicity, either by its export or degradation. Therefore we chose to look more closely at predicted urea carboxylase genes (Kanamori et al., 2004), which are frequently controlled by guanidine-I riboswitches (Figure 1B). Urea carboxylases and ureases have been proposed to catalyze the two major routes for the biological decomposition of urea. However, this redundancy seems unnecessary, and it has been previously hypothesized that one of these protein families might have an alternative purpose (Hausinger, 2004). Intriguingly, urea carboxylase genes are commonly associated with guanidine-I riboswitches, whereas ureases never appear to be controlled by this riboswitch class. Therefore, we speculated that urea carboxylases might prefer guanidine as a substrate (Figure 5A) and initiate its degradation in a pathway that is similar to that currently proposed for urea.

Figure 5. Guanidine-related Functions for Protein Products of Genes Associated with Guanidine-I Riboswitches.

Figure 5

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(A) Proposed reaction scheme for the carboxylation of guanidine by an enzyme previously proposed to function as a urea carboxylase (Kanamori et al., 2004).

(B) Plot of the velocities of a previously defined urea carboxylase versus the concentration of guanidine or urea. Data points are the average of three technical replicates.

(C) List of the enzymatic properties of a carboxylase enzyme with guanidine and urea substrates. Values shown are the averages and standard deviations of three independent experiments.

(D) Phylogenetic tree showing the genetic variability within SMR transporters controlled by guanidine-I riboswitches. Selected experimentally validated EmrE proteins that are known not to be controlled by the riboswitch are shown in purple.

(E) Recognition of guanidine by predicted SMR transporters controlled by guanidine riboswitches evaluated by tryptophan emission spectra of Clo SugE at pH 7.5 in the presence of increasing guanidine.

(F) Plot of the increase in fluorescence of tryptophan residues within Clo SugE at 333 nm versus the concentration of guanidine. Background fluorescence in the absence of guanidine is subtracted from each data point. Solid line represents the fit to a 1-to-1 binding model with a KD of 1 mM. Error bars indicate the standard error of the experiment. When not visible, error bars are smaller than the data point.

(G) Plot of the intrinsic fluorescence of tryptophan residues within Clo SugE at 333 nm versus the concentration of guanidine in the presence and absence of 1 mM TPP+. Other annotations are as described for F. Data are the average of three measurements. Error bars indicate the standard error of the experiment. When not visible, error bars are smaller than the data point.

The only previously characterized prokaryotic urea carboxylase, from the Proteobacterium Oleomonas sagaranensis, is encoded by a gene located immediately downstream of a mini-ykkC RNA. As noted above, we speculate that guanidine-I riboswitches and the noncoding mini-ykkC and ykkC-III motif RNAs might have similar gene control functions. The O. sagaranensis urea carboxylase indeed can use both urea and guanidine as substrates (Figure 5B). Importantly, the KM for urea is in the millimolar range (7.2 mM reported by Kanamori et al., 2004; 5.2 mM observed in this study, Figure 5C), whereas the KM for guanidine improves to 0.19 mM. Thus, the enzyme carboxylates guanidine with a catalytic efficiency roughly 40-fold better than with urea. Since the maximum velocity and kcat are only marginally higher compared to urea, this increased efficiency is presumably due to the enhanced affinity of the enzyme for guanidine.

Urea carboxylases are thought to have been introduced to eukaryotes by a horizontal gene transfer event from Proteobacteria (Strope et al., 2011). An x-ray structural model of the carboxylase domain of the urea amidolyase from the eukaryote Kluyveromyces lactis has been solved previously (Fan et al., 2012). Based on this structure, it is hypothesized that a protonated aspartic acid residue near the active site, which is conserved in the O. sagaranensis carboxylase, forms a hydrogen bond with the oxygen atom of urea. For this to be true, however, the side-chain carboxylic acid of aspartate would have to undergo a pKa shift of nearly 4 units. In contrast, this residue could more easily form an ionic interaction or a hydrogen bond with guanidine without undergoing a shift in pKa. This mechanism would explain the apparent enhanced affinity for guanidine over urea as described above. These observations suggest that diverse species of bacteria and eukarya express carboxylase enzymes that have evolved to degrade guanidine.

A Riboswitch-controlled Transport Protein Selectively Binds Guanidine

One of the most intriguing aspects of the guanidine-I riboswitch class is its association with genes predicted to code for small multidrug resistance (SMR) proteins. This family is subdivided into small multidrug proteins (SMP), suppressor of groEL mutation (SUG) proteins, and paired small multidrug resistance (PSMR) proteins. The latter group includes the proteins YkkC and YkkD in B. subtilis (Bay et al., 2008), which are regulated by a guanidine-I riboswitch (Figures 2 and S6). To date, most studies have been performed either on the SMP E. coli EmrE protein, which has been found to transport a wide variety of positively charged small molecules (Bay and Turner, 2012; Yerushalmi et al., 1995), or on the E. coli SugE protein, which has been suggested to transport considerably fewer compounds (Chung and Saier Jr., 2002).

To explore how SMR proteins encoded by genes controlled by guanidine-I riboswitches differ from the well-studied E. coli EmrE transporter, we constructed a phylogenetic tree of these proteins (Figure 5D). The riboswitch almost always controls a pair of SMR proteins, one of which appears to be EmrE-like and the other SugE-like. Very frequently, their genes are found in a single operon, which suggests that heterodimers of EmrE-like and SugE-like proteins might form naturally. Additionally, when a guanidine-I riboswitch controls only a single copy of an emrE- or sugE-like gene, its putative partner is always found elsewhere in the genome, most often controlled by another guanidine riboswitch. In rare instances, guanidine-I riboswitches also control a single SMR gene that is highly similar to E. coli sugE. In contrast, genes that are highly similar to E. coli emrE are never controlled by the riboswitch.

We speculated that SMR proteins whose expression is controlled by guanidine-I riboswitches might naturally function as guanidine transporters. In a preliminary investigation of this hypothesis, we examined a representative SugE protein (called Clo SugE) that is derived from a Clostridiales bacterium (oral taxon 876) and controlled by a guanidine-I riboswitch. The distribution of positive charges on the loops suggested that this protein was likely to assemble as a homo-complex (Rapp et al., 2006), and size exclusion chromatography of the purified protein supported this inference. A dose-dependent increase in the intrinsic fluorescence of tryptophan residues within the Clo SugE protein was observed upon the addition of guanidine, demonstrating that it binds guanidine (Figure 5E). The KD for this interaction is ~1 mM, as estimated by fitting the fluorescence data to a single-site binding model (Figure 5F). Importantly, a similar urea titration has no effect on Clo SugE fluorescence, and guanidine titration has no effect on an unrelated fluoride transporter with a similar number of tryptophan residues (data not shown).

Finally, we sought to determine whether Clo SugE was capable of selectively recognizing guanidine by monitoring the affinity of their interaction in the presence of 1 mM tetraphenylphosphonium (TPP+), a typical substrate of the E. coli EmrE protein. A concentration of TPP+ at ~1,000-fold higher than its KD for an E. coli EmrE (Morrison and Henzler-Wildman, 2014) fails to disrupt guanidine binding (Figure 5G). These results suggest that this riboswitch controlled SMR protein selectively recognizes guanidine and not the bulky, cationic substrates known to be transported by distal members of this general family of proteins.

Free Guanidine is Naturally Produced by Bacteria

The findings described above demonstrate the existence of riboswitches that selectively respond to free guanidine, and suggest that proteins are widely produced by bacteria that degrade and transport guanidine. However, there are no previous reports regarding the natural production of guanidine at biologically-relevant concentrations (Mitchell, 1985, 1987). It seems unlikely that an ancient riboswitch and its associated super-regulon would respond to an unnatural compound. Therefore, we speculated that guanidine might be produced through natural metabolic processes under certain conditions, and that its presence had not yet been detected, perhaps due to its small size or its perceived irrelevance. Accordingly, we sought to identify conditions where a guanidine riboswitch reporter is expressed even though no external guanidine is supplied.

If bacteria experience conditions under which guanidine is produced, the compound might be ejected from cells into the growth medium. Therefore, an E. coli strain that lacks the tolC gene was chosen to examine our hypothesis because the TolC protein is a well-known exit channel for small toxic molecules in Gram-negative bacteria (Koronakis, 2003). Indeed the ΔtolC strain, but not the WT strain, exhibits robust WT riboswitch reporter gene expression when grown overnight in minimal medium (Figure 6A). The M4 construct (Figure S3A), which carries a single mutation in the aptamer domain, exhibits lower induction by guanidine as expected. These data suggest that the riboswitch ligand is synthesized by E. coli under these conditions, and accumulates in cells that are deficient in exporting small toxic compounds.

Figure 6. Natural Production of Free Guanidine by Bacteria.

Figure 6

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(A) E. coli WT and tolC knockout (ΔtolC) cells carrying the K. pneumoniae WT or M4 riboswitch reporter constructs (Figure S3) grown in rich (LB) or minimal (GMM) liquid media.

(B) Plots of the mass-to-charge ratios (m/z) of compounds with similar retention times to authentic guanidine and authentic Boc-guanidine. Asterisks identify peaks from the extract LC-MS spectrum within 10 ppm of the calculated mass of each authentic compound as designated.

(C) LC-MS analysis for guanidine from the ΔtolC strain of E. coli grown in minimal medium as depicted in A. Extract and authentic guanidine samples were analyzed directly or were treated with Boc-anhydride to alter the mobility and mass of free guanidine as noted.

Upon analyzing the contents of these bacteria via high-resolution liquid chromatography-mass spectrometry (LC-MS), we observed the presence of a compound with a mass-to-charge ratio equivalent to that of guanidine (Figure 6B). This compound also had a similar retention time to that of guanidine (Figure 6C). We speculated that the modest difference in these retention times might be due to the intrinsic differences of a pure guanidine solution and a complex extract sample. To further confirm that this compound was, in fact, guanidine, we treated cell extracts with di-tert-butyl dicarbonate (Boc-anhydride) to convert guanidine into Boc-guanidine. A new compound with the identical mass-to-charge ratio and similar retention time as authentic Boc-guanidine was observed. Upon mixing the Boc-anhydride-treated extract and Boc-guanidine, a single LC-MS peak was observed (Figure 6C), indicating that these peaks both correspond to Boc-guanidine. These results demonstrate that E. coli is capable of endogenously producing guanidine.

DISCUSSION

It has been proposed that the cognate ligands for widespread orphan riboswitch classes are likely to be involved in underappreciated, but fundamental, aspects of biology (Breaker, 2011). The data described in the current report supports the conclusion that free guanidine is the natural ligand for the predominant subtype of ykkC motif RNAs, the most common and longest unresolved orphan riboswitch candidate. However, we should note that it remains possible that guanidine-I riboswitches might have some additional function that would contribute to the extensive sequence conservation observed in this motif. Despite being generated over 150 years ago, only in rare instances has guanidine been considered to be a biologically relevant compound. However, our results (Figure 6) demonstrate that guanidine is naturally produced by bacteria. Perhaps many bacteria generate free guanidine as they utilize suboptimal energy sources when preferred sources such as glucose are in short supply.

Guanidine-I riboswitches, and the structurally unrelated mini-ykkC and ykkC-III motif RNAs (Weinberg et al., 2010) that likely function as guanidine riboswitches (Breaker Laboratory, data not shown), control a super-regulon that includes genes for proteins that appear to detoxify free guanidine by degradation or export. Given the widespread distribution of guanidine riboswitches in bacteria, free guanidine might have been biologically relevant in early life forms. Notably, guanidine is one of a number of compounds that could possibly have been part of a prebiotic pathway for purine synthesis (Becker et al., 2016). Guanidine degradation in bacteria could occur in the same manner proposed for urea, wherein previously annotated urea carboxylase enzymes preferentially use guanidine as a substrate (Figure 5A). Genes coding for such ‘guanidine carboxylases’ are almost always found associated with two families of uncharacterized proteins. It seems likely that these other proteins play some additional role in guanidine metabolism. Notably, the product of carboxylation of guanidine, carboxyguanidine, is a close analog of allophanate. Therefore, proteins annotated as allophanate hydrolases whose expression levels are controlled by guanidine riboswitches presumably use carboxyguanidine as a substrate for subsequent decomposition.

Another class of genes commonly regulated by these riboswitches encode arginases, which typically catalyze the conversion of arginine into ornithine. Examination of the guanidine riboswitches that are upstream of the genes encoding these arginases reveals that they most likely activate gene expression in the presence of guanidine. When controlled by the riboswitch, these genes are always found in an operon with the genes hypA and hypB, which have been implicated in the insertion of Ni2+ into hydrogenases in E. coli (Lutz et al., 1991). Since arginases can also utilize a Ni2+ ion at their active site (Viator et al., 2008), we speculate that hypA and hypB may be involved in the insertion of Ni2+ or some other cation into these enzymes. Perhaps guanidine-induced expression of this operon helps cells overcome inhibition of arginase activity by free guanidine. Alternatively, these enzymes could conceivably catalyze the difficult reverse reaction, where ornithine or 5-hydroxynorvaline is directly converted to arginine via the addition of guanidine.

Guanidine riboswitches also commonly control the expression of numerous transporters. Many of these are predicted to import nitrate, sulfate, or bicarbonate. It seems possible that these proteins might transport bicarbonate to facilitate the carboxylation of guanidine and its subsequent metabolism. Indeed, genes encoding such transporters are often found in an operon with guanidine carboxylases genes. The majority of the remaining transporters whose expression levels are controlled by guanidine riboswitches are predicted SMR transporters. Certain members of this protein superfamily, such as the E. coli proteins EmrE and SugE have been extensively studied as model transporters due to their small size. EmrE in particular has been demonstrated to transport a wide variety of positively charged small molecules ranging from TPP+ to glycine betaine (Bay and Turner, 2012; Yerushalmi et al., 1995). We demonstrated that one of the SMR proteins whose expression is controlled by a guanidine riboswitch selectively recognizes guanidine. Our data further suggest that SMR transporters whose expression levels are controlled by guanidine-I riboswitches usually assemble as heterodimers of SugE- and EmrE-like subunits and that their biological role is to function as guanidine exporters. This hypothesis is supported by our observation that B. subtilis cells wherein this transporter is knocked out have a greater sensitivity to guanidine (Figure 2D) and a higher cellular concentration of the riboswitch ligand (Figure 2E and 2F).

In E. coli, the SugE protein is controlled by the mini-ykkC motif, suggesting it might also transport guanidine. Interestingly, the sugE gene was first identified as a suppressor of a groEL mutant gene, a gene that encodes one of a family of molecular chaperones that assists in the proper folding of proteins (Greener et al., 1993). An intriguing possibility is that SugE reduces the cellular concentration of guanidine, a known protein destabilization agent, thereby reducing the need for GroEL to chaperone protein folding.

It is also important to note that emrE genes not controlled by guanidine-I riboswitches are commonly associated with horizontal gene transfer elements and are relatively recently diverged (Bay et al., 2008). In contrast, genes that encode SMR proteins that are controlled by these RNAs are only rarely found within 10 kb of an integrase or transposase. These observations suggest that a mutation may have occurred in an ancestral guanidine transporter that allowed it to transport a wider variety of cationic compounds, leading to its rapid transfer amongst bacteria and subsequent contribution to antibiotic resistance.

As noted earlier, approximately 25% of ykkC motif RNAs conform to subtype 2 (Figure S1A). Members of this subtype are commonly associated with genes necessary for de novo purine metabolism and branched-chain amino acid biosynthesis. These RNAs are distinguished by nucleotide variations at the sites where the structure of guanidine-I riboswitches modulates in the presence of guanidine. Therefore, subtype 2 ykkC motif RNAs appear to have adapted to recognize one or more unknown ligands that relate to the metabolic processes relevant to their associated genes. Future experiments will be required to establish the ligand(s) for this remaining collection of orphan riboswitches.

EXPERIMENTAL PROCEDURES

Bioinformatics

Additional homologs of ykkC motif RNAs were identified using Infernal 1.1 (Nawrocki and Eddy, 2013), as described in the Extended Experimental Procedures. The hits were separated based on nucleotide sequences into subtype 1 (~1500 sequences) (Figure 1A) and subtype 2 (Figure S1A) ykkC motif RNAs (~500 sequences) using R2R (Weinberg and Breaker, 2011). Genes predicted to be controlled by guanidine riboswitches were determined as described previously (Nelson et al., 2013). Construction of a phylogenetic tree containing riboswitch-controlled and experimentally studied SMR proteins is described in the Extended Experimental Procedures.

Riboswitch Reporter Assays and Knockout Strain Preparation

Riboswitch reporter fusion constructs were created via PCR of the riboswitch sequence of interest with DNA primers carrying either its native promoter or the lysC promoter from B. subtilis or E. coli as appropriate, followed by restriction digestion and cloning into either the pDG1661 or pRS414 vector. Constructs subsequently were used to transform B. subtilis str. 168 1A1 or E. coli BW25113 as indicated for each experiment. Gene knockouts were constructed via insertion of the tet cassette into the gene of interest via homologous recombination. Reporter expression was analyzed using 4-methylumbelliferyl β-D-galactopyranoside as described previously (Nelson et al., 2015). Further experimental details can be found in the Extended Experimental Procedures.

Agar Diffusion Assays

B. subtilis strains carrying riboswitch-reporter constructs were spread on LB agar plates containing X-gal (100 μg mL−1) and appropriate antibiotics. Autoclaved 6 mm diameter paper discs prepared from 0.35 mm thick pure cellulose chromatography paper (Fisher Scientific) were soaked with 10 μL of compound at specific concentrations and transferred to the prepared agar plates. The plates were incubated overnight at 37°C.

In-line Probing Assays

Methods for the preparation of RNA constructs are described in the Extended Experimental Procedures. In-line probing assays were conducted as described previously (Regulski and Breaker, 2008; Soukup and Breaker, 1999). Briefly, 5′ 32P-labeled RNAs were incubated at room temperature with candidate ligands in 20 mM MgCl2, 100 mM KCl, 50 mM Tris [pH 8.3 at 23°C] for 40 hours. The resulting spontaneous RNA cleavage products were separated by denaturing (8 M urea) 10% polyacrylamide gel electrophoresis (PAGE) and were visualized and quantified by using a Typhoon phosphorimager (GE Healthcare). Dissociation constants were established by varying the concentration of ligand added and quantifying the changes in intensity of spontaneous RNA cleavage bands that are altered by ligand-induced structural modulation. Band intensity was then normalized to a non-modulating band, the resulting values were scaled between 0 and 1, and the values were plotted versus the logarithm of the ligand concentration. Values for apparent dissociation constants (KD) were determined using a sigmoidal-dose response equation and GraphPad Prism 6.

Single-round Transcription Termination Assays

Detailed procedures for single-round transcription termination assays can be found in the Extended Experimental Procedures. Briefly, DNA constructs for in vitro transcription were designed to include the promoter of the pfl gene from Clostridium beijerinckii, the riboswitch aptamer and expression platform of interest, and sufficient nucleotides following the terminator stem to distinguish between full length and terminated constructs via denaturing PAGE. DNA constructs were transcribed in the presence or absence of various concentrations of guanidine and the amount of full-length and terminated RNA elucidated by quantifying the phosphorimager data collected after PAGE. Values for the fraction of full-length RNA transcripts were calculated as previously described (Kim et al., 2007).

Guanidine Carboxylase Expression and Enzymatic Assays

The full-length urea carboxylase (uca) gene from O. sagaranensis containing a His6 tag at its N-terminus was cloned into the pETDuet-1 expression vector (Novagen). The modified protein was expressed in E. coli BL21(DE3) cells containing the expression vector and purified by nickel-affinity followed by strong anion exchange chromatography. Analysis of the catalytic activity of the purified protein was performed as described previously (Kanamori et al., 2004). Additional details can be found in the Extended Experimental Procedures.

SugE Binding Assays

E. coli Bl21-DE3 cells were transformed with a pET21a+ plasmid bearing a synthetic gene insertion encoding a riboswitch-associated small multidrug resistance (SMR) protein from Clostridiales bacterium oral taxon 876 (NCBI reference sequence WP_021653285.1), containing a His6 tag at its C-terminus. The modified protein was expressed and purified via cobalt affinity chromatography followed by gel filtration. Fluorescence experiments were carried out with 5 micromolar protein in 150 mM NaCl, 25 mM HEPES (pH 7.5 at 23°C), 5 mM n-decyl-β-D-maltopyranoside (DM), and increasing amounts of guanidine hydrochloride. Additional details can be found in the Extended Experimental Procedures.

LC-MS Analysis of Bacterial Cell Extracts

E. coli containing a deletion in the tolC gene were grown with shaking at 37°C overnight in GMM minimal medium in the absence of guanidine. A cell extract was prepared and analyzed via high-resolution LC-MS using an Agilent 6550 qToF mass spectrometer. Data were analyzed using the Agilent Mass Hunter software. Peaks were considered to have the same mass-to-charge ratio as guanidine or Boc-guanidine if they were within 10 ppm of the calculated ratio. Further experimental details can be found in the Extended Experimental Procedures.

Supplementary Material

Acknowledgments

We thank Adam Roth, Zasha Weinberg, Narasimhan Sudarsan, and other members of the Breaker Lab for helpful conversations. We also thank Shira Stav for assistance in assembling the dataset of RNA sequences, Sanshu Li for experimental reagents, and Christina Lünse and Takemasa Kawashima for assistance with literature translations. We are grateful to Christopher Lim, Joshua Temple, and Yong Xiong of Yale University for assistance in the expression and purification of the guanidine carboxylase protein, and Rob Bjornson for assisting our use of the Yale Life Sciences High Performance Computing Center (NIH grant RR19895-02). Finally, we thank the Bacillus Genomic Stock Center at The Ohio State University and the Coli Genetics Stock Center at Yale University for supplying bacterial strains and plasmids. R.M.A. was supported by an NIH T32 Genomic and Proteomics Training Grant (3T32HG003198-10S2). M.E.S. and C.W.R were supported by an NIH Cellular and Molecular Biology Training Grant (T32GM007223). This work was also supported by NIH grants to R.B.S. (R00-GM111767) and to R.R.B. (GM022778 and DE022340). R.R.B. is also supported by the Howard Hughes Medical Institute.

Footnotes

SUPPLEMENTAL INFORMATION

Supplemental Information includes Extended Experimental Procedures, 12 figures, one table and one data file and can be found with this article online at.

AUTHOR CONTRIBUTIONS

JWN undertook the ligand screening campaign, and JWN, RMA, MES and RRB contributed to the experimental design and/or data analysis of genetic and biochemical studies on riboswitches and guanidine carboxylase. RBS contributed data on SMR protein phylogeny and biochemistry. JWN and RRB were responsible for creating drafts of the manuscript with assistance from all authors.

References

  1. Barrick JE, Corbino KA, Winkler WC, Nahvi A, Mandal M, Collins J, Lee M, Roth A, Sudarsan N, Jona I, et al. New RNA motifs suggest an expanded scope for riboswitches in bacterial genetic control. Proc Natl Acad Sci USA. 2004;101:6421–6426. doi: 10.1073/pnas.0308014101. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bay DC, Rommens KL, Turner RJ. Small multidrug resistance proteins: a multidrug transporter family that continues to grow. Biochim Biophys Acta. 2008;1778:1814–1838. doi: 10.1016/j.bbamem.2007.08.015. [DOI] [PubMed] [Google Scholar]
  3. Bay DC, Turner RJ. Small multidrug resistance protein EmrE reduces host pH and osmotic tolerance to metabolic quaternary cation osmoprotectants. J Bacteriol. 2012;194:5941–5948. doi: 10.1128/JB.00666-12. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Becker S, Thoma I, Deutsch A, Gehrke T, Mayer P, Zipse H, Carell T. A high-yielding, strictly regioselective prebiotic purine nucleoside formation pathway. Science. 2016;352:833–836. doi: 10.1126/science.aad2808. [DOI] [PubMed] [Google Scholar]
  5. Bierema S. Die assimilation von ammon-, nitrat- und amidstickstoff durch mikroorganismen. Centr Bakt Par. 1909;2:672–726. [Google Scholar]
  6. Block KF, Hammond MC, Breaker RR. Evidence for widespread gene control function by the ydaO riboswitch candidate. J Bacteriol. 2010;192:3983–3989. doi: 10.1128/JB.00450-10. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Breaker RR. Prospects for riboswitch discovery and analysis. Mol Cell. 2011;43:867–879. doi: 10.1016/j.molcel.2011.08.024. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Chung YJ, Saier MH., Jr Overexpression of the Escherichia coli sugE gene confers resistance to a narrow range of quaternary ammonium compounds. J Bacteriol. 2002;184:2543–2545. doi: 10.1128/JB.184.9.2543-2545.2002. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Dambach M, Sandoval M, Updegrove TB, Anantharaman V, Aravind L, Waters LS, Storz G. The ubiquitous yybP-ykoY riboswitch is a manganese-responsive regulatory element. Mol Cell. 2015;57:1099–1109. doi: 10.1016/j.molcel.2015.01.035. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Dann CE, 3rd, Wakeman CA, Sieling CL, Baker SC, Irnov I, Winkler WC. Structure and mechanism of a metal-sensing regulatory RNA. Cell. 2007;130:878–892. doi: 10.1016/j.cell.2007.06.051. [DOI] [PubMed] [Google Scholar]
  11. Ebisuno T, Takimoto M. Examination on the biodegradability of guanyl compounds. Eisei Kagaku. 1981;27:156–162. [Google Scholar]
  12. Fan C, Chou CY, Tong L, Xiang S. Crystal structure of urea carboxylase provides insights into the carboxyltransfer reaction. J Biol Chem. 2012;287:9389–9398. doi: 10.1074/jbc.M111.319475. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Greene RF, Pace CN. Urea and guanidine hydrocholoride denaturation of ribonuclease, lysozyme, α-chymotrypsin, and β-lactoglobulin. J Biol Chem. 1974;249:5388–5393. [PubMed] [Google Scholar]
  14. Greener T, Govezensky D, Zamir A. A novel multicopy supressor of a groEL mutation includes two nested open reading frames transcribed from different promoters. EMBO J. 1993;12:889–896. doi: 10.1002/j.1460-2075.1993.tb05729.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Hausinger RP. Metabolic versatility of prokaryotes for urea decomposition. J Bacteriol. 2004;186:2520–2522. doi: 10.1128/JB.186.9.2520-2522.2004. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Gusarov I, Nudler E. The mechanism of intrinsic transcription termination. Mol Cell. 1999;3:495–504. doi: 10.1016/s1097-2765(00)80477-3. [DOI] [PubMed] [Google Scholar]
  17. Hermann T, Westhof E. Docking of cationic antibiotics to negatively charged pockets in RNA folds. J Med Chem. 1999;42:1250–1261. doi: 10.1021/jm981108g. [DOI] [PubMed] [Google Scholar]
  18. Iwanoff NN, Awetissowa AN. Über die fermentative umwandlung des guanidins in harnstoff. Biochem Z. 1931;231:67–78. [Google Scholar]
  19. Jack DL, Storms ML, Tchieu JH, Paulsen IT, Saier MH., Jr A broad-specificity multidrug efflux pump requiring a pair of homologous SMR-type proteins. J Bacteriol. 2000;182:2311–2313. doi: 10.1128/jb.182.8.2311-2313.2000. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Kanamori T, Kanou N, Atomi H, Imanaka T. Enzymatic characterization of a prokaryotic urea carboxylase. J Bacteriol. 2004;186:2532–2539. doi: 10.1128/JB.186.9.2532-2539.2004. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Kihara H, Prescott JM, Snell EE. The bacterial cleavage of canavanine to homoserine and guanidine. J Biol Chem. 1955;217:497–503. [PubMed] [Google Scholar]
  22. Kim JN, Roth A, Breaker RR. Guanine riboswitch variants from Mesoplasma florum selectively recognize 2′-deoxyguanosine. Proc Natl Acad Sci USA. 2007;104:16092–16097. doi: 10.1073/pnas.0705884104. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Kim PB, Nelson JW, Breaker RR. An ancient riboswitch class in bacteria regulates purine biosynthesis and one-carbon metabolism. Mol Cell. 2015;57:317–328. doi: 10.1016/j.molcel.2015.01.001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Koronakis V. TolC – the bacterial exit duct for proteins and drugs. FEBS Lett. 2003;555:66–71. doi: 10.1016/s0014-5793(03)01125-6. [DOI] [PubMed] [Google Scholar]
  25. Lutz S, Jacobi A, Schlensog V, Böhm R, Sawers G, Böck A. Molecular characterization of an operon (hyp) necessary for the activity of the three hydrogenase isoenzymes in Escherichia coli. Mol Microbiol. 1991;5:123–135. doi: 10.1111/j.1365-2958.1991.tb01833.x. [DOI] [PubMed] [Google Scholar]
  26. Mandal M, Lee M, Barrick JE, Weinberg Z, Emilsson GM, Ruzzo WL, Breaker RR. A glycine-dependent riboswitch that uses cooperative binding to control gene expression. Science. 2004;306:275–279. doi: 10.1126/science.1100829. [DOI] [PubMed] [Google Scholar]
  27. Marchais A, Duperrier S, Durand S, Gautheret D, Stragier P. CsfG, a sporulation-specific, small non-coding RNA highly conserved in endospore formers. RNA Biol. 2011;8:358–364. doi: 10.4161/rna.8.3.14998. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Marescau B, Nagels G, Possemiers I, De Broe ME, Becaus I, Billiouw JM, Lornov W, De Deyn PP. Guanidino compounds in serum and urine of nondialyzed patients with chronic renal insufficiency. Metabolism. 1997;46:1024–1031. doi: 10.1016/s0026-0495(97)90273-0. [DOI] [PubMed] [Google Scholar]
  29. Meyer MM, Hammond MC, Salinas Y, Roth A, Sudarsan N, Breaker RR. Challenges of ligand identification for riboswitch candidates. RNA Biol. 2011;8:5–10. doi: 10.4161/rna.8.1.13865. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Mitchell WR. Biodegradation of guanidinium by aquatic microorganisms. US Army Med Res and Dev Command Tech Rep. 1985;8506:1–26. [Google Scholar]
  31. Mitchell WR. Biodegradation of guanidinium ion in aerobic soil samples. Bull Environ Contam Toxicol. 1987;39:974–981. doi: 10.1007/BF01689587. [DOI] [PubMed] [Google Scholar]
  32. Morrison EA, Henzler-Wildman KA. Transported substrate determines exchange rate in the multidrug resistance transporter EmrE. J Biol Chem. 2014;289:6825–6836. doi: 10.1074/jbc.M113.535328. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Natelson S, Sherwin JE. Proposed mechanism for urea nitrogen re-utilization: relationship between urea and proposed guanidine cycles. Clin Chem. 1979;25:1343. [PubMed] [Google Scholar]
  34. Nawrocki EP, Eddy SR. Infernal 1.1: 100-fold faster RNA homology searches. Bioinformatics. 2013;29:2933–2935. doi: 10.1093/bioinformatics/btt509. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Nelson JW, Plummer MS, Blount KF, Ames TD, Breaker RR. Small molecule fluoride toxicity agonists. Chem Biol. 2015;22:527–534. doi: 10.1016/j.chembiol.2015.03.016. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Nelson JW, Sudarsan N, Furukawa K, Weinberg Z, Wang JX, Breaker RR. Riboswitches in eubacteria sense the second messenger c-di-AMP. Nat Chem Biol. 2013;9:834–839. doi: 10.1038/nchembio.1363. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Price IR, Gaballa R, Ding F, Helmann JD, Ke A. Mn2+-sensing mechanisms of yybP-ykoY orphan riboswitches. Mol Cell. 2015;57:1110–1123. doi: 10.1016/j.molcel.2015.02.016. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Pyle AM. Metal ions in the structure and function of RNA. J Biol Inorg Chem. 2002;7:679–690. doi: 10.1007/s00775-002-0387-6. [DOI] [PubMed] [Google Scholar]
  39. Rapp M, Granseth E, Seppala S, von Heijne G. Identification and evolution of dual-topology membrane proteins. Nat Struct Mol Biol. 2006;13:112–116. doi: 10.1038/nsmb1057. [DOI] [PubMed] [Google Scholar]
  40. Regulski EE, Breaker RR. In-Line Probing Analysis of Riboswitches. Methods Mol Biol. 2008;419:53–67. doi: 10.1007/978-1-59745-033-1_4. [DOI] [PubMed] [Google Scholar]
  41. Reiss C, Strobel SA. Submitted 2016 [Google Scholar]
  42. Roth A, Winkler WC, Regulski EE, Lee BW, Lim J, Jona I, Barrick JE, Ritwik A, Kim JN, Welz R, et al. A riboswitch selective for the queuosine precursor preQ1 contains an unusually small aptamer domain. Nat Struct Mol Biol. 2007;14:308–317. doi: 10.1038/nsmb1224. [DOI] [PubMed] [Google Scholar]
  43. Schuldiner SS. EmrE, a model for studying evolution and mechanism of ion-coupled transporters. Biochim Biophys Acta. 2009;1794:748–762. doi: 10.1016/j.bbapap.2008.12.018. [DOI] [PubMed] [Google Scholar]
  44. Schuldiner SS. Undecided membrane proteins insert in random topologies. Up, down and sideways: it does not really matter. Trends Biochem Sci. 2012;37:215–219. doi: 10.1016/j.tibs.2012.02.006. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Sekowska A, Danchin A, Risler JL. Phylogeny of related functions: the case of polyamine biosynthetic enzymes. Microbiology. 2000;146:1815–1828. doi: 10.1099/00221287-146-8-1815. [DOI] [PubMed] [Google Scholar]
  46. Serganov A, Nudler E. A decade of riboswitches. Cell. 2013;152:17–24. doi: 10.1016/j.cell.2012.12.024. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Soukup GA, Breaker RR. Relationship between internucleotide linkage geometry and the stability of RNA. RNA. 1999;5:1308–1325. doi: 10.1017/s1355838299990891. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Strecker A. Untersuchungen über die chemischen Beziehungen zwischen Guanin, Xanthin, Theobromin, Caffeïn und Kreatinin. Justus Liebigs Ann Chem. 1861;118:151–177. [Google Scholar]
  49. Strope PK, Nickerson KW, Harris SD, Moriyama EN. Molecular evolution of urea amidolyase and urea carboxylase in fungi. BMC Evol Biol. 2011;11:80. doi: 10.1186/1471-2148-11-80. 2011. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Viator RJ, Rest RF, Hildebrandt E, McGee DJ. Characterization of Bacillus anthracis arginase: effects of pH, temperature, and cell viability on metal preference. BMC Biochem 2008. 2008 doi: 10.1186/1471-2091-9-15. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Walker JB, Walker MS. The enzymatic reduction of hydroxyguanidine. J Biol Chem. 1959;234:1481–1484. [PubMed] [Google Scholar]
  52. Weinberg Z, Barrick JE, Yao Z, Roth A, Kim JN, Gore J, Wang JX, Lee ER, Block KF, Sudarsan N, et al. Identification of 22 candidate structured RNAs in bacteria using the CMfinder comparative genomics pipeline. Nucleic Acids Res. 2007;35:4809–4819. doi: 10.1093/nar/gkm487. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Weinberg Z, Breaker RR. R2R – software to speed the depiction of aesthetic consensus RNA secondary structures. BMC Bioinfo. 2011;12:3. doi: 10.1186/1471-2105-12-3. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Weinberg Z, Wang JX, Bogue J, Yang J, Corbino K, Moy RH, Breaker RR. Comparative genomics reveals 104 candidate structured RNAs from bacteria, archaea, and their metagenomes. Genome Biol. 2010;11:R31. doi: 10.1186/gb-2010-11-3-r31. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Winkler WC, Nahvi A, Roth A, Collins JA, Breaker RR. Control of gene expression by a natural metabolite-responsive ribozyme. Nature. 2004;428:281–286. doi: 10.1038/nature02362. [DOI] [PubMed] [Google Scholar]
  56. Yarnell WS, Roberts JW. Mechanism of intrisnic transcription termination and antitermination. Science. 1999;284:611–615. doi: 10.1126/science.284.5414.611. [DOI] [PubMed] [Google Scholar]
  57. Yerushalmi H, Lebendiker M, Schuldiner S. EmrE, an Escherichia coli 12-kDa multidrug transporter, exchanges toxic cations, and H+ and is soluble in organic solvents. J Biol Chem. 1995;270:6856–6863. doi: 10.1074/jbc.270.12.6856. [DOI] [PubMed] [Google Scholar]

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