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. 2018 Nov 2;46(19):10082-10094.
doi: 10.1093/nar/gky709.

A Glutamine Riboswitch Is a Key Element for the Regulation of Glutamine Synthetase in Cyanobacteria

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Free PMC article

A Glutamine Riboswitch Is a Key Element for the Regulation of Glutamine Synthetase in Cyanobacteria

Stephan Klähn et al. Nucleic Acids Res. .
Free PMC article

Abstract

As the key enzyme of bacterial nitrogen assimilation, glutamine synthetase (GS) is tightly regulated. In cyanobacteria, GS activity is controlled by the interaction with inactivating protein factors IF7 and IF17 encoded by the genes gifA and gifB, respectively. We show that a glutamine-binding aptamer within the gifB 5' UTR of Synechocystis sp. PCC 6803 is critical for the expression of IF17. Binding of glutamine induced structural re-arrangements in this RNA element leading to enhanced protein synthesis in vivo and characterizing it as a riboswitch. Mutagenesis showed the riboswitch mechanism to contribute at least as much to the control of gene expression as the promoter-mediated transcriptional regulation. We suggest this and a structurally related but distinct element, to be designated type 1 and type 2 glutamine riboswitches. Extended biocomputational searches revealed that glutamine riboswitches are exclusively but frequently found in cyanobacterial genomes, where they are primarily associated with gifB homologs. Hence, this RNA-based sensing mechanism is common in cyanobacteria and establishes a regulatory feedback loop that couples the IF17-mediated GS inactivation to the intracellular glutamine levels. Together with the previously described sRNA NsiR4, these results show that non-coding RNA is an indispensable component in the control of nitrogen assimilation in cyanobacteria.

Figures

Figure 1.
Figure 1.
Occurrence of the RF01739 (glnA aptamer) and the RF01704 (DP aptamer) motifs among cyanobacterial genomes. The phylogenetic tree was generated using the Neighbor-Joining method (55) and the 16S rDNA sequences of 60 representative cyanobacteria. Evolutionary analyses were conducted in MEGA7 (56). The RNA motif frequency within a particular genome is color coded. If the frequency was >3 the exact number of motifs was specified. Furthermore, the proteins encoded downstream of both RNA motifs were identified using the Pfam database. Note that DUF4278 is part of the GS inactivating factor IF17. Thus, the Pfam hits referring to DUF4278 most likely represent IF17 homologs or IF17-like proteins. If the number of Pfam hits was lower than the number of reported RNA motif hits, then not every downstream query returned a hit in the Pfam database. A detailed description is provided in the Material and Methods section. The complete dataset, i.e. the exact loci of the individual RNA motifs are given in Supplementary Table S1.
Figure 2.
Figure 2.
The gifB 5′ UTR of Synechocystis harbors a glutamine-binding aptamer. (A) Predicted secondary structure of the glnA aptamer upstream of the gifB gene. The nucleotide positions refer to the transcriptional start site of the gifB gene (+1, ref. (21)). R1–3 label the regions for which structural modulation was observed (see panel B). The secondary structure is based on the secondary structure model of glnA motif RNAs published previously (42). (B) In-line probing analysis using a 5′ 32P-labeled gifB 5′ UTR. Precursor RNA (Pre) was incubated for 2 days at 23°C without a ligand (0) or in presence of increasing concentrations of L-glutamine (3.2 μM to 10 mM, half-log dilutions). The same RNA was also partially treated with Na2CO3, which mediates alkaline degradation (OH) or with RNase T1, which cleaves after G residues (T1). A non-treated RNA served as control (NR, no reaction). (C) Densitometric evaluation of the bands corresponding to regions R1–3 whose intensities changed due to the presence of glutamine (marked by red lines in panel B). The intensities were normalized to bands which did not show glutamine-dependent intensity changes (Ctrl). (D) In-line probing using mutated RNAs. The NR, T1 and OH samples are shown for the WT RNA.
Figure 3.
Figure 3.
GFP fluorescence measurements of Synechocystis strains carrying translational fusions of DUF4278-encoding genes from Synechocystis or Prochlorococcus sp. MED4 with the superfolder gfp gene. (A) Schematic illustration of the reporter constructs on a pVZ322 plasmid. The construct representing the glnA aptamer contained the entire 5′ UTR of gifB and 54 nt of its coding region in frame with the sfgfp gene. Transcription was driven by the BioBrick BBa_J23101 promoter (iGEM Registry, (48)) which was found to be constitutively active at a high level in Synechocystis (49). (B) Raw fluorescence of a WT (no sfgfp gene present) and a reporter strain carrying the Ctrl plasmid shown in panel A. Both strains had the same OD750. For each following measurement the signals measured from a simultaneously cultivated WT was subtracted from the measured fluorescence values (background correction). (C–E) Background corrected GFP fluorescence of strains carrying the control construct (Ctrl, C) or point mutated versions of the glnA aptamer (D, E). Please note that M3 is the complementary mutation to M1 (see Figure 2A). At time point 0, 10 mM NH4Cl (f.c.) were added to the cultures (indicated by arrow). In parallel, the same number of independent cultures remained untreated (negative control, n.c.). (F) Background corrected GFP fluorescence of reporter strains under standard (non-induced) growth conditions. (G) Background corrected GFP fluorescence of a Synechocystis strain carrying a DP aptamer within the 5′ UTR of the gene WP_071812974 of Prochlorococcus sp. MED4 in fusion with sfgfp (as shown in panel A). The measurement was performed under standard growth conditions and after adding 20 mM NH4Cl. Data are the mean ± SD of three biological replicates.
Figure 4.
Figure 4.
Mutagenesis of the glutamine riboswitch reveals its importance for gifB expression and GS regulation. (A) Schematic overview of the mutated genomic locus in Synechocystis strains carrying chromosomal point mutations in the glutamine riboswitch upstream of gifB. The kanamycin resistance (KmR) gene was introduced between the promoters of gifB and slr1597. The gifB::Ctrl strain harbors the WT sequences whereas in gifB::M1 and gifB::M3 the glutamine-binding aptamer carries point mutations M1 or M3 as shown in Figure 2A. Red arrows indicate the relative binding sites of primers used for PCR (panel B). RS - recombination site, chr. - chromosome. (B) PCR verification of the mutant strains using two biological replicates. Using WT gDNA a 977 bp fragment was amplified whereas a fragment of 2032 bp was amplified in the mutants. The point mutations M1 and M3 were verified by sequencing the PCR product. No WT allele was amplified in the mutants indicating that they are fully segregated. (C) Immunoblots showing the NH4+ induced accumulation of IF17 (upper band). At time point 0, 10 mM NH4Cl (f.c.) were added to cells grown in BG11 containing nitrate as sole N source. The TrxA protein level is shown as loading control (lower band). (D) Kinetics of intracellular glutamine levels in response to an upshift in NH4+ availability (same conditions and treatment as used for immunoblots). Data are the mean of the two biological replicates.
Figure 5.
Figure 5.
Simplified overview of N control and GS regulation in the cyanobacterial model strain Synechocystis involving two different types of non-coding RNAs. AMT - ammonium transport system, NRT - nitrate transport system, CM – cell membrane, NaR - nitrate reductase, NiR - nitrite reductase, GS – glutamine synthetase, Gln - glutamine, Glu – glutamate, 2OG - 2-oxoglutarate, Gln-RS – glutamine riboswitch.

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