A nitrogen stress-inducible small RNA regulates CO2 fixation in Nostoc

Abstract

In the absence of fixed nitrogen, some filamentous cyanobacteria differentiate heterocysts, specialized cells devoted to fixing atmospheric nitrogen (N2). This differentiation process is controlled by the global nitrogen regulator NtcA and involves extensive metabolic reprogramming, including shutdown of photosynthetic CO2 fixation in heterocysts, to provide a microaerobic environment suitable for N2 fixation. Small regulatory RNAs (sRNAs) are major post-transcriptional regulators of gene expression in bacteria. In cyanobacteria, responding to nitrogen deficiency involves transcribing several nitrogen-regulated sRNAs. Here, we describe the participation of nitrogen stress-inducible RNA 4 (NsiR4) in post-transcriptionally regulating the expression of two genes involved in CO2 fixation via the Calvin cycle: glpX, which encodes bifunctional sedoheptulose-1,7-bisphosphatase/fructose-1,6-bisphosphatase (SBPase), and pgk, which encodes phosphoglycerate kinase (PGK). Using a heterologous reporter assay in Escherichia coli, we show that NsiR4 interacts with the 5'-untranslated region (5'-UTR) of glpX and pgk mRNAs. Overexpressing NsiR4 in Nostoc sp. PCC 7120 resulted in a reduced amount of SBPase protein and reduced PGK activity, as well as reduced levels of both glpX and pgk mRNAs, further supporting that NsiR4 negatively regulates these two enzymes. In addition, using a gfp fusion to the nsiR4 promoter, we show stronger expression of NsiR4 in heterocysts than in vegetative cells, which could contribute to the heterocyst-specific shutdown of Calvin cycle flux. Post-transcriptional regulation of two Calvin cycle enzymes by NsiR4, a nitrogen-regulated sRNA, represents an additional link between nitrogen control and CO2 assimilation.

Figure 1

NsiR4 in Nostoc. A, Schematic representation of the region encoding NsiR4. The flanking annotated open reading frames are indicated (gray arrows), together with NsiR4 (orange arrow) and the promoter region cloned upstream of the gfp gene (green arrow) in promoter-probe plasmid pELV71. B, Sequence of the promoter and coding region of NsiR4. The sequence encoding NsiR4 (in orange), the stop codon of alr3725 (in red), as well as the NtcA-binding box, the −10 box, the spacing between both boxes (22 nt), and the TSS (black arrow) are indicated. Sequences producing a stem-loop terminator are indicated with orange arrows. C and D, Nitrogen-responsive expression of NsiR4 in Nostoc WT (C) and hetR mutant strain 216 (D). Expression was analyzed by Northern blot in cells grown in the presence of NH₄⁺ and transferred to a medium containing no source of combined nitrogen for the number of hours indicated. The upper panels show hybridization to a probe for NsiR4. The lower panels show hybridization to a probe for 5S RNA used as loading and transfer control. Samples contained 10 µg of total RNA.

Figure 2

Expression of P_nsiR4-gfp. A, Confocal fluorescence images of filaments bearing plasmid pELV71 growing on top of medium containing ammonium (NH₄⁺) or lacking any source of combined nitrogen (N₂) are shown with merged green (GFP fluorescence) and red (autofluorescence, shown in magenta) channels. Quantification of the signal for the green channel along the filaments indicated with white arrows is shown on the right. All images were acquired with the same sensitivity settings for the green channel so that intensities can be compared. Scale bars, 10 µm. B, Quantification of GFP fluorescence in vegetative cells of the filaments indicated with white arrows in (A). Sixty three regions of interest corresponding to vegetative cells were quantified for each condition (red ovals), as indicated in the scheme on the left. Data are presented on the right as the mean and standard deviation of the fluorescence. T test ****P <0.0001.

Figure 3

Verification of NsiR4 interaction with the 5′-UTR of glpX and pgk using an in vivo reporter system in E. coli. A, Left, predicted interaction between NsiR4 and the 5′-UTR of glpX according to IntaRNA (Mann et al., 2017). Nucleotides in the 5′-UTR are numbered with respect to the start of the coding sequence (shaded). The mutation introduced in NsiR4 at position 13 (C to G, Mut13) and the corresponding compensatory mutation in glpX 5′-UTR at position −20 (G to C, Comp13) are indicated in blue and purple, respectively. Hybridization energies of the interaction between the glpX mRNA and the WT (black) or mutated (blue) versions of NsiR4 are indicated. Right, fluorescence measurements of E. coli DH5α cultures bearing combinations of plasmids expressing different versions of NsiR4 (WT or Mut13) and glpX::sfgfp fusions (WT or Comp13). Plasmid pJV300 was used as control. B, Left, predicted interaction between NsiR4 and the 5′-UTR of pgk according to IntaRNA (Mann et al., 2017). Nucleotides in the 5′-UTR are numbered with respect to the start of the coding sequence (shaded). The mutation introduced in NsiR4 at position 13 (C to G, Mut13) is indicated in blue. Hybridization energies of the interaction between the pgk mRNA and the WT (black) or mutated (blue) versions of NsiR4 are indicated. Right, fluorescence measurements of E. coli DH5α cultures bearing combinations of plasmids expressing different versions of NsiR4 (WT or Mut13) and the pgk::sfgfp fusion. Plasmid pJV300 was used as control. A and B, Putative SD sequences are boxed. The data are presented as the mean ± standard deviation of the results from eight independent colonies after subtraction of fluorescence in cells bearing plasmid pXG0. Fluorescence is normalized to the A₆₀₀ of each culture. T test ***P <0.001; *P <0.05.

Figure 4

Effect of NsiR4 on the amount of SBPase protein, PGK activity, and the accumulation of glpX and pgk mRNAs in Nostoc. A, Scheme of the DNA fragments cloned into the plasmids used to generate the different strains with altered levels of NsiR4 (orange arrows). TSS (bent arrows), Rho-independent terminator of NsiR4 (small stem-loop), T1 terminator (large stem-loop) and the trc promoter are indicated. B, Northern blot using RNA extracted from strains OE_C, OE_NsiR4, and OE_as_NsiR4 grown in the presence of nitrate (0) or 24 h after nitrogen removal (24), hybridized with a probe for NsiR4 (top) or 5S RNA (bottom) used as loading control. Endogenous NsiR4 (black triangles) and the 6-nt-longer NsiR4 version constitutively expressed from the trc promoter (red triangle) are indicated. Samples contained 10 µg of total RNA. C, Accumulation of SBPase in strains with altered levels of NsiR4 was analyzed by Western blot in samples containing 40 µg of soluble fraction from cells grown in the presence of nitrate and transferred to medium containing no source of combined nitrogen for 24 h. Western blots were quantified with ImageLab software (Bio-Rad, Hercules, CA, USA) and normalized to Ponceau Red staining. The position of size markers (in kilodalton) is indicated on the right side. Four technical replicates of two different clones of each strain were quantified. Values are expressed as percentage of the normalized SBPase amount with respect to the amount in the control strain (OE_C), which was considered 100%. D, PGK activity in strains with altered levels of NsiR4 was measured in crude extracts from cells analyzed in (C). Assays were performed in duplicate with two different clones of each strain. Specific activity is expressed as percentage of the activity (2.1 U/mg protein) in the control strain (OE_C), which was considered 100%. E and F, Accumulation of glpX (E) or pgk (F) mRNAs in strains with altered levels of NsiR4 were analyzed by Northern blot in the same cells analyzed in (C and D). The position of size markers (in kilobase) is indicated on the left side. Data from two different clones of each strain were quantified and the amount of glpX or pgk mRNA was normalized to the amount of rnpB RNA. Values are expressed as percentage of the glpX or pgk RNA amount, normalized with respect to RNA amount in the control strain (OE_C), which was considered 100%. Samples contained 3.5 µg of total RNA. C–F, The data are presented as the mean ± standard deviation. T test *P <0.05; ^**P <0.01; ***P <0.001.

Figure 5

Schematic representation of the enzymatic reactions carried out by SBPase (in blue) and PGK (in orange) in the context of the Calvin–Benson cycle, together with the regulatory effects described here for NsiR4. The differential accumulation of NsiR4 in heterocysts versus vegetative cells is indicated with different font size and solid versus dashed red lines. Previously described regulation of SBPase by a heterocyst-specific antisense RNA (Olmedo-Verd et al., 2019) is also indicated. Gray crosses on PRK and Rubisco highlight that these enzymes are absent in heterocysts (Codd and Stewart, 1977; Codd et al., 1980; Cossar et al., 1985). 1,3-BPG, 1,3-bisphosphoglycerate; 3PG, 3-phosphoglycerate; DHAP, dihydroacetone phosphate; E4P: eritrose-4-phosphate; F-1,6-BP, fructose-1,6-bisphosphate; F6P, fructose-6-phosphate; G3P, glyceraldehide-3-phosphate; R5P, ribose-5-phosphate; Ru-1,5-BP, ribulose-1,5-bisphosphate; Ru5P, ribulose-5-phosphate; S1,7-BP, sedoheptulose-1,7-bisphosphate; S7P, sedoheptulose-7-phosphate; X5P, xilulose-5-phosphate. The reduced operation of CO₂ fixation in heterocysts versus vegetative cells is indicated by gray arrows.