Na+ riboswitches regulate genes for diverse physiological processes in bacteria

Abstract

Organisms presumably have mechanisms to monitor and physiologically adapt to changes in cellular Na⁺ concentrations. Only a single bacterial protein has previously been demonstrated to selectively sense Na⁺ and regulate gene expression. Here we report a riboswitch class, previously called the 'DUF1646 motif', whose members selectively sense Na⁺ and regulate the expression of genes relevant to sodium biology. Many proteins encoded by Na⁺-riboswitch-regulated genes are annotated as metal ion transporters, whereas others are involved in mitigating osmotic stress or harnessing Na⁺ gradients for ATP production. Na⁺ riboswitches exhibit dissociation constants in the low mM range, and strongly reject all other alkali and alkaline earth ions. Likewise, only Na⁺ triggers riboswitch-mediated transcription and gene expression changes. These findings reveal that some bacteria use Na⁺ riboswitches to monitor, adjust and exploit Na⁺ concentrations and gradients, and in some instances collaborate with c-di-AMP riboswitches to coordinate gene expression during osmotic stress.

Fig. 1. The DUF1646 motif associates with genes related to metal cation transport.

a, Consensus sequence and secondary structure model of the riboswitch candidate class called the DUF1646 motif. The consensus model is based on the alignment of 308 nonredundant examples. b, Existing annotations of the protein products whose genes are associated with DUF1646 motif RNAs. c, Alkali (I) and alkaline earth (II) cations considered as candidate ligands for the putative aptamer formed by DUF1646 motif RNAs. Numbers represent the ionic radius (pm) in crystal form for the mono- or divalent forms of the elements indicated.

Fig. 2. Selective binding of Na⁺ by a natural RNA aptamer.

a, Sequence and secondary structure of the WT 66 kefB RNA construct used to assess ligand-binding characteristics of a DUF1646 motif RNA. The two lowercase letters designate guanosine nucleotides appended to the natural bacterial sequence from the kefB gene to facilitate efficient production by in vitro transcription. Mutant constructs M1 and M2 carry the boxed nucleotide changes at the sites indicated. Circles identify positions of notable strand scission from the in-line probing data depicted in b. b, PAGE separation of product bands resulting from in-line probing assay reactions reveal RNA shape changes induced by the addition of Na⁺. 5′ ³²P-labelled precursor (Pre) RNA was subjected to no reaction (NR), partial digestion with RNase T1 (T1) (cleaves after G nucleotides), incubation in alkali conditions (^–OH), or incubation in in-line probing reactions with buffer alone (including 2 mM MgCl₂ but lacking KCl) (−) or supplemented with various amounts of Na⁺ ranging from 10 μM to 100 mM. Bands corresponding to some products generated by RNase T1 digestion are identified by the guanosine nucleotide position located immediately 5′ of the cleavage site. Bands of interest were mapped according to their sites of spontaneous strand scission on the RNA construct depicted in a. c, Plot of the fraction of RNA bound to Na⁺ as estimated from the in-line probing data depicted in b. Band intensities at sites 1 and 2 were used to generate the plot. The line depicts an idealized binding curve for a 1-to-1 interaction and a K_D of 2.2 mM. d, PAGE analysis of in-line probing assays using 5′ ³²P-labelled 66 kefB RNA and conducted in the presence of various alkali metal cations or NH₄⁺ at 10 mM. e, PAGE analysis of in-line probing assays using 5′ ³²P-labelled WT 66 kefB RNA or the mutant M1 or M2 versions of this construct. In-line probing reactions were conducted in the absence (−) or presence (+) of 10 mM Na⁺.

Fig. 3. Na⁺ selectively triggers riboswitch-mediated transcription read-through and increased gene expression.

a, WT Na⁺ riboswitch model based on the DUF1646 motif representative from the C. acetobutylicum kefB gene. Approximate 3′ termini for the terminated (T) and FL RNA transcripts are indicated. FL carries an additional 60 nucleotides (encircled number) not shown. The genetic reporter construct carries different 3′ nucleotides, including the lacZ gene. Additional annotations are as describe for Figs. 1 and 2. b, Top, representative PAGE analysis of in vitro transcription termination assays. Bands corresponding to the T and FL RNA products as defined in a are indicated. Lane M (marker) was loaded with a transcript corresponding to FL RNA. Bottom, a plot of the fraction of FL transcripts versus ion supplementation for various constructs. Data points (n = 3) corresponding to the PAGE autoradiogram depicted are coloured the same as the annotation panels. Open and black-filled data points were derived from two additional replicate assays (autoradiograms not shown). Solid lines represent average values for each condition, and the dashed line represents the average value for the WT construct with no Na⁺ supplementation. All transcription reactions include at least 4 mM Na⁺ due to its presence in the standard transcription reaction buffer, and this is not reflected in the ion identities and concentrations presented in the graphic. c, Representative reporter assays using surrogate B. subtilis cells carrying the riboswitch–reporter construct depicted in a. Cells were cultured in low-sodium LB media at the indicated pH value and containing roughly 15 mM Na⁺ and X-gal, which was used either without alteration (–) or was supplemented with 150 mM NaCl (Extended Data Fig. 5 and Supplementary Table 1). See Methods for additional details. d, Representative reporter assays conducted as described in c using WT or M4 riboswitch reporters cultured in low-sodium LB medium at pH 9, used without alteration (–) or supplementation with the monovalent ions indicated (Extended Data Fig. 5 and Supplementary Table 1). Lower Li⁺ concentration was used to avoid strong growth inhibition. Source data

Fig. 4. A natural tandem arrangement between riboswitches for Na⁺ and c-di-AMP operates as a two-input Boolean logic gate.

a, Sequence and secondary structure model for the tandem riboswitch system associated with the lysine-2,3-aminomutase gene of Dehalobacter sp. CF. b, Top, a truth table for an A and Not B (material nonimplication) Boolean logic gate. The expected (exp.) output represents the gene expression outcome for an individual RNA where the riboswitches operate perfectly. Green represents active gene expression. Bottom, the expected gene expression trends as influenced by biological conditions. c, Top, in vitro transcription assays demonstrating independent operation of each riboswitch. T1, T2 and FL identify bands corresponding to RNAs ending at terminator 1, terminator 2, or reaching full length, respectively. Other annotations are as described for Fig. 3b. Bottom, a plot of the fraction of total transcripts terminating at sites T1, T2 and FL as depicted in the gel lanes above. Values were established by measuring band intensities and adjusting for the number of radiolabelled U nucleotides (Methods). d, Plots of the total fraction of in vitro transcription products proceeding past T1 (top) or T2 (bottom). Values from replicate experiments (n = 3) are represented. Solid lines represent the average values for each condition and gray lines represent the average value in the absence of Na⁺ and c-di-AMP inputs for comparison. Source data

Extended Data Fig. 1. Na⁺-mediated structural modulation of a DUF1646 motif RNA representative from Lactococcus garvieae Lg2.

a, Sequence and secondary structure model for the 53 mgtA construct derived from L. garvieae Lg2. Annotations are as described for Fig. 2a, with the in-line probing data derived from b. b, PAGE analysis of in-line probing reactions with trace amounts of 5′ ³²P-labelled 53 mgtA RNA. Annotations are as described for Fig. 2b. c, Plot of the estimated fraction of RNA bound to Na⁺ generated by monitoring band intensity changes at sites 1 and 2 as depicted in b. The line depicts an idealized binding curve for a 1-to-1 interaction between RNA and Na⁺, yielding a K_D of ~15 mM. Additional annotations are as described for Fig. 2c.

Extended Data Fig. 2. The 66 kefB RNA retains Na⁺ binding ability in the presence of 100 mM K⁺.

a, Sequence and secondary structure model of the 66 kefB RNA construct with mapped in-line probing data generated in the presence of various Na⁺ concentrations. Data was derived from the assay results depicted in b. Additional annotations are as described for Fig. 2a. b, PAGE separation of product bands resulting from in-line probing assay reactions conducted using the 66 kefB RNA construct incubated with 0 to 100 mM NaCl. Additional annotations are as described for Fig. 2b except that 100 mM KCl was also present in each reaction. c, Plot of the fraction of RNA bound to Na⁺ as estimated from the in-line probing data depicted in b. Additional annotations are as described for Fig. 2c.

Extended Data Fig. 3. DUF1646 motif RNAs reject alkaline earth divalent cations.

PAGE separation of product bands from in-line probing assays wherein 5′ ³²P-labelled 66 kefB RNA was incubated with various alkaline earth divalent cations. Annotations are as described for Fig. 2b. Note that only the Na⁺ control assay (includes 10 mM NaCl and 2 mM MgCl₂) yields the banding pattern changes indicative of ligand binding. The other assays depicted were conducted with 2 mM MgCl₂ supplemented with 10 mM of the divalent metal ions indicated, and these do not yield the same banding pattern changes exclusively observed upon the addition of Na⁺. Also note that some metal ions such as Mg²⁺ enhance the overall rate of spontaneous RNA cleavage as expected due to their ability to favor deprotonation of the RNA 2′ hydroxyl group.

Extended Data Fig. 4. Ligand-binding specificity of a DUF1646 motif representative from Thermoanaerobacterium thermosaccharolyticum M0795.

a, Sequence and secondary structure model for a DUF1646 representative from T. thermosaccharolyticum M0795 carrying 66 nucleotides and including two nonnative G nucleotides (gg) on the 5′ terminus to facilitate efficient production by in vitro transcription. b, Autoradiogram of PAGE-separated products resulting from in-line probing assays using the RNA construct depicted in a. In-line probing reactions were modified from the standard reactions conditions to exclude KCl and to contain low (2 mM) MgCl₂. Monovalent ions as annotated for each lane were added to the reaction mixture at either 500 mM (Li⁺, Na⁺, Cs⁺, NH₄⁺), 300 mM (K⁺), or 50 mM (Rb⁺). Divalent ions were tested at 10 mM. Notes: (i) Some divalent ions such as Mg²⁺ accelerate RNA strand scission in a concentration-dependent manner, and therefore these ions were not tested at higher concentrations. (ii) The lane with Sr²⁺ appears to be loaded with less RNA, which results in a broad reduction of intensities of all bands. Regardless, the lack of selective band modulation suggests that Sr²⁺ is not specifically bound by the RNA.

Extended Data Fig. 5. Quantitation of riboswitch-reporter fusion assays.

B. subtilis cells carrying the WT or M4 riboswitch-reporter constructs depicted in Fig. 3a were grown overnight in low-sodium (~15 mM Na⁺) LB (yeast extract and tryptone). 20 µL of the overnight culture was subcultured in low-sodium LB buffered at pH 7.0 (100 mM PIPES), pH 8.0 (100 mM TAPS), or pH 9.0 (100 mM AMPSO), all containing chloramphenicol (5 µg/mL). As indicated for each assay, ions were added to supplement the media, the mixtures were incubated overnight, and ONPG (ortho-nitrophenyl-ß-galactoside) was added to samples to measure β-galactosidase activity (units presented in Supplementary Table 1) by adapting the method described previously [Miller, J.H. Procedures for working with lac. In: A Short Course in Bacterial Genetics. (Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press) pp 72 (1992)]. Three independent replicates of bacterial cultures and gene expression assays were performed for each condition. Depicted are plots of gene expression normalized by setting the low Na⁺ β-galactosidase activity units to 1 (dashed line). Red lines represent the mean for the three independent experiments. See Materials and Methods for additional details. Notes: Li⁺ and Mg²⁺ concentrations were chosen to avoid strong growth inhibition. Due to the poor solubility of Ca²⁺, Sr²⁺, and Ba²⁺ at pH 9.0, 1 mM concentrations were used because 10 mM concentrations in LB were observed to precipitate.

Extended Data Fig. 6. Three Na⁺ riboswitches in Acetivibrio cellulolyticus regulate three major processes related to sodium biology.

The bacterium A. cellulolyticus carries three Na⁺-I riboswitch representatives (red) that regulate genes associated with three major aspects of sodium biology. Sodium gradient: The pycB gene codes for oxaloacetate decarboxylase, whose activity drives Na⁺ export to yield a Na⁺ gradient [Dimroth, P., Jockel, P. & Schmid, M. Coupling mechanism of the oxaloacetate decarboxylase Na⁺ pump. Biochim. Biophys. Acta Bioenerg. 1505, 1-14 (2001)]. The gradient can then be exploited to promote solute uptake by Na⁺-dependent import proteins [Pos, K. M. & Dimroth, P. Functional properties of the purified Na⁺-dependent citrate carrier of Klebsiella pneumoniae: evidence for asymmetric orientation of the carrier protein in proteoliposomes. Biochemistry 35, 1018-1026 (1996)]. pH homeostasis: DUF1646 genes code for proteins of unknown function but some have been predicted to function as a Na⁺/H⁺ antiporters [CDD Conserved Protein Domain Family: DUF1646 (nih.gov); https://www.genome.jp/dbget-bin/www_bget?pop:7465038], which presumably can be used to increase the pH of cells growing under alkaline conditions. Osmotic stress response: Tandem Na⁺-I and c-di-AMP (brown) riboswitches are associated with dapB, which codes for dihydrodipicolinate reductase. This enzyme produces 2,3,4,5-tetrahydrodiaminopimelate, which is a precursor for both lysine and peptidoglycan biosynthesis [https://www.genome.jp/pathway/lin00300+dapB]. Peptidoglycan biosynthesis is critical for cell resistance to osmotic stress in high salt environments [Auer, G. K. & Weibel, D. B. Bacterial cell mechanics. Biochemistry 56, 3710-3724 (2017); Metris, A., George, S. M., Mulholland, F., Carter, A. T. & Baranyi, J. Metabolic shift of Escherichia coli under salt stress in the presence of glycine betaine. Appl. Environ. Microb. 80, 4745-4756 (2014)]. E. coli cells grown in 4.5% NaCl exhibit increased expression of dapB, and our findings indicate that some species directly regulate this gene using a Na⁺-responsive tandem riboswitch.