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A Mn-sensing riboswitch activates expression of a Mn2+/Ca2+ ATPase transporter in Streptococcus

Abstract Maintaining manganese (Mn) homeostasis is important for the virulence of numerous bacteria. In the human respiratory pathogen Streptococcus pneumoniae, the Mn-specific importer PsaBCA, exporter MntE, and transcriptional regulator PsaR establish Mn homeostasis. In other bacteria, Mn homeostasis is controlled by yybP-ykoY family riboswitches. Here, we characterize a yybP-ykoY family riboswitch upstream of the mgtA gene encoding a PII-type ATPase in S. pneumoniae, suggested previously to function in Ca2+ efflux. We show that the mgtA riboswitch aptamer domain adopts a canonical yybP-ykoY structure containing a three-way junction that is compacted in the presence of Ca2+ or Mn2+ at a physiological Mg2+ concentration. Although Ca2+ binds to the RNA aptamer with higher affinity than Mn2+, in vitro activation of transcription read-through of mgtA by Mn2+ is much greater than by Ca2+. Consistent with this result, mgtA mRNA and protein levels increase ≈5-fold during cellular Mn stress, but only in genetic backgrounds of S. pneumoniae and Bacillus subtilis that exhibit Mn2+ sensitivity, revealing that this riboswitch functions as a failsafe ‘on’ signal to prevent Mn2+ toxicity in the presence of high cellular Mn2+. In addition, our results suggest that the S. pneumoniae yybP-ykoY riboswitch functions to regulate Ca2+ efflux under these conditions.


Supplementary Materials and Methods
Bacterial strain and plasmid construction. Bacterial strains and plasmids used in this study are listed in Table S1 and relevant DNA oligonucleotide primers in Table S2. All Streptococcus pneumoniae D39 serotype 2 mutants are derivatives of strain IU1781 (wild-type). S. pneumoniae null-mutants were constructed by gene deletion replacement and counter antibiotic selection using the rpsL + cassette, Janus, as previous described (1,2). S. pneumoniae strains expressing C-terminal triple-FLAG-tagged MgtA from its native promoter were constructed by joining overlapping fragments generated by PCR to the Pc-erm R resistance marker. A 10 amino acid linker (5'-ggttccgctggctccgctgctggttctggc-3') was inserted before the triple-FLAG tag (5'gattataaagatgatgatgataaagattataaagatgatgatgataaagattataaagatgatgatgataaataa-3'; stop codon underlined) generating mgtA-L-FLAG 3 -Pc-erm R . When needed, antibiotics were added at the following concentrations (µg mL -1 ): kanamycin, 250; streptomycin, 250; and erythromycin 0.3.
For single-copy promoter-lacZ fusions, oligonucleotide primers designed with restriction sites were used to amplify the mgtA (spd1383) promoter region, including its 5'-UTR region and the first 12 amino acids of the coding region of mgtA from S. pneumoniae genomic DNA. Truncated variations were also constructed where indicated. Amplicons were cloned into the pDG1663 vector and transformed into Escherichia coli DH5α cells (3). Resulting plamid constructs were confirmed by restriction analysis and DNA sequence verified. Plasmids were linearized by XhoI and transformed into Bacillus subtilis 168 strain CU1065 (wild-type) and the null-mntR mutant strain, where they integrated into the thrC locus (3). Site-directed mutagenesis was used to generate nucleotide substitutions in the 5'-UTR region of the mgtA riboswitch on plasmid pJEM1663d before integrating into B. subtilis strains as described above. When needed, antibiotics were added to media at the following concentrations (µg mL -1 ): for E. coli, ampicillin, 50 or 100; for B. subtilis, erythromycin, 1; lincomycin, 25; and spectinomycin, 100.
For construction of B. subtilis strains overexpressing MgtA or its riboswitch aptamer domain, oligonucleotide primers were used to amplify selected target region from S. pneumoniae genomic DNA. Amplicons were cloned into pPL82 vector under the Pspac(hy) promoter (4) by restriction digest-ligation or by gibson isothermal assembly methods and transformed into E. coli DH5α cells. The resulting plasmid constructs were confirmed by restriction analysis and sequence verified. Plasmids were linearized by PstI, transformed, and integrated into the amyE locus of B. subtilis strains. When needed, antibiotics were added to media at the following concentrations (µg mL -1 ): for E. coli, chloramphenicol, 30; for B. subtilis, chloramphenicol, 10; and ampicillin, 100.
Bacterial growth. S. pneumoniae strains were routinely cultured in brain heart infusion (BHI) broth at 37°C in an atmosphere of 5% CO2. Briefly, bacteria glycerol stocks were inoculated into BHI broth, serially diluted, and grown overnight. The next day, exponentially growing overnight cultures were diluted into BHI to 0.005 OD620 with or without metal stress as indicated and growth was followed overtime. Bacterial cultures were harvested at 3.5 h for most experiments unless indicated. B. subtilis strains were routinely cultured in Luria-Bertani (LB) broth at 37°C with agitation. Isopropyl β-D-1-thiogalactopyranoside (IPTG) was added at 1 mM to medium when needed to induce expression of target gene.
Disk diffusion assay. Susceptibility to Mn was tested by mixing 100 µL of mid-exponential phase LB cultures with 4 mL of LB top agar (0.8 % agar) and poured onto solidified LB agar plates. Sterile paper discs (6 mm) impregnated with increasing concentrations of MnCl2 were placed on top of the agar and plates were incubated at 37°C. The diameter of the zone of inhibition was measured at 24 h. IPTG was added at 1 mM in both the top agar and agar plates for B. subtilis strains overexpressing MgtA or the mgtA riboswitch aptamer. β-galactosidase activity assay. B. subtilis strains were grown to 0.15 OD600 in LB, treated with or without metals as indicated. After 2 h incubation at 37°C, cells were collected by centrifugation, lysed, and β-galactosidase activity was measured according to Miller, 1972 (5).
Quantitative real-time PCR. Total RNA with enrichment for transcripts <200 nucleotides was extracted using the combination of the miRNeasy kit (Qiagen) and the lyse matrix B tubes (MP biomedicals). Briefly, bacterial cell pellets from 2 to 4 ml of culture were suspended in 700 µL Qiazol lysis reagent, transferred to a lyse matrix B tube, and lysed by a beadbeater. RNA extraction was continued according to the manufacturer's protocol for the miRNAeasy kit. On the column DNA digestion was performed with RNase-free DNAse 27.3 Kunitz unit (Qiagen). Eluted RNA was converted to cDNA (QuantaBio). PCR amplification was carried out using oligonucleotide primers listed in Table S3. PCR outcomes were normalized to the house keeping gene of gyrA or 16s rRNA for S. pneumoniae or B. subtilis samples, respectively. Relative transcript levels were calculated by comparison of the ratio of treated to non-treated wild-type cells.
Western blot. Protein expression was determined from S. pneumoniae strains harboring chromosomal fusions of C-terminal triple-FLAG tagged MgtA protein grown in BHI with or without 200 µM MnCl2 for approximately 3.5 h at 37°C in an atmosphere of 5% CO2. Cells were centrifuged, washed with 1x phosphate buffered saline [pH 7.4], lysed, and 20 µg total proteins were separated by SDS-PAGE. Proteins were transferred to a nitrocellulose membrane (Biorad). Membranes were blocked with ECL blocking agent (GE healthcare) and probed with anti-FLAG rabbit polyclonal antibody (Sigma). Target protein was detected using anti-rabbit antibody linked to horseradish peroxidase (Sigma) and ECL western blotting reagent (GE healthcare), then imaged using a ChemiDoc imaging system (Boirad). yybP-ykoY RNA synthesis. The S. pneumoniae D39 DNA wild-type mgtA 5' UTR (containing the 99-nt yybP-ykoY aptamer domain) sequence was cloned into pUC19 following a T7 RNA polymerase promoter. The first 5' CG base-pair was converted to GC base-pair to support in vitro transcription using an in-house purified T7 RNA polymerase. The DNA template for in vitro transcription was amplified by PCR using primers listed in Table S2. Site-directed mutagenesis using two partially complemented primers was used to generate nucleotide substitutions in the parent plasmid construct (6). All plasmids constructed were sequence verified.
Inductively coupled plasma-mass spectrometry (ICP-MS) for measurement of total cellassociated metal. The total amounts of cell-associated Mn, Fe, Co, Ni, Zn, Cu, or Ca were quantified from 5-20 mL bacterial cultures as described for each experiment. Cells were centrifuged, washed once with ice-cold phosphate buffered saline (PBS) [pH 7.4] containing 2 mM nitrilotriacetic acid or ethylenediaminetetraacetic acid (EDTA), washed twice with ice-cold metal-free PBS (10g/L chelex-100 resin was used to remove metals; Biorad) [pH 7.4], and dried for 2 h using a centrifuge evaporator. Dried cells were solubilized in 400 μL 30% (v/v) HNO3 and lysed by incubating at 95°C for 10 min shaking at 500 rpm. ICP-MS samples were prepared by diluting 300 μL of lysed cells into 2.7 mL 2.5% (v/v) HNO3. Metal concentrations were calculated from the standard curve using 1 to 50 ppb metal stock solutions and normalized to total protein determined using DC TM protein assay (Biorad).   (8), and the figure was prepared using Jalview (9).  RNAs. Folding of WT (B) and mutant A18C (G) constructs were quantified using imageJ (11). E, extended form; C, compact form. The relative intensities of the extended (E) and compact (C) conformations (band fractions) were fit using a single site binding model with DynaFit (12) to determine Mn 2+ binding affinities (apparent dissociation equilibrium constants, Kd) for wild-type (WT) and the C18A mutant. The Mn 2+ -bound was assumed to be exclusively in the C conformation; the Mn 2+ binding constant and the E-C equilibrium in the absence of Mn 2+ ions were allowed to vary in the fit (50% E for WT, 90% E for the C18A mutant). Average fraction ± SD of C (black circles) and E (red circles) are shown from three independent experiments.        S12. S. pneumoniae MgtA shares sequence similarity with secretory pathway Ca 2+ ATPases (SPCAs) known to transport both Mn 2+ and Ca 2+ . Multiple-sequence analysis of S. pneumoniae D39 MgtA protein aligned with SPCAs previously identified in yeast and vertebrates and known to transport both Mn 2+ and Ca 2+ into the Golgi from the cytosol (13)(14)(15). Amino acids with 100 percent identity are highlighted red; amino acids involved in metal transport are highlighted ochre; amino acids that bind Ca 2+ (in the crystal structure of Oryctolagus cuniculus SERCA1) are highlighted green; and the residue Q747 proposed to affect metal selectivity is highlighted magenta. Secondary structure of O. cuniculus SERCA1is shown below the amino acid sequence, where α-helices are red and β-sheets are blue. The following sequences were used for this alignment: Homo sapiens SPCA1; L. monocytogenes LMCA1; O. cuniculus SERCA1; and S. pneumoniae D39 spd_1383. The alignment was generated using Kalign (8), and the figure was prepared using Jalview (9).   The following gblock gene fragment sequence was used to construct the Pr_term lacZ fusion: GAGATAGATTGCTTCATTGAATTCCCTTTCTAGTCACATAGCATTCTACCATTATACTGGTTT TCAAGGAAAAATTCAAATAAGGCAGTTTTTACTCTTTGAAAAGACCCCAAAATTTGGTATAAT GACTTGTTATGATTAACAGGTCTCTTTTTGTTTGTCGTGAAAAGATATGACAGAAAGAGAGT CTGTTTTGCACACAATGTCAAGGAGGAGACACATGTCAAAAGAACAAAAACGCCAAGCGTT TTATACTCGGATCCCTGAAGAG; predicted terminator hairpin (gray) of the mgtA riboswitch is preceded by the 100 ntd promoter sequence; restriction sites are italicized. Table S3. qRT-PCR primers used in this study.