The Nitrogen Stress-Repressed sRNA NsrR1 Regulates Expression of all1871, a Gene Required for Diazotrophic Growth in Nostoc sp. PCC 7120

Small regulatory RNAs (sRNAs) are post-transcriptional regulators of bacterial gene expression. In cyanobacteria, the responses to nitrogen availability, that are mostly controlled at the transcriptional level by NtcA, involve also at least two small RNAs, namely NsiR4 (nitrogen stress-induced RNA 4) and NsrR1 (nitrogen stress-repressed RNA 1). Prediction of possible mRNA targets regulated by NsrR1 in Nostoc sp. PCC 7120 allowed, in addition to previously described nblA, the identification of all1871, a nitrogen-regulated gene encoding a protein of unknown function that we describe here as required for growth at the expense of atmospheric nitrogen (N2). We show that transcription of all1871 is induced upon nitrogen step-down independently of NtcA. All1871 accumulation is repressed by NsrR1 and its expression is stronger in heterocysts, specialized cells devoted to N2 fixation. We demonstrate specific interaction between NsrR1 and the 5′ untranslated region (UTR) of the all1871 mRNA, that leads to decreased expression of all1871. Because transcription of NsrR1 is partially repressed by NtcA, post-transcriptional regulation by NsrR1 would constitute an indirect way of NtcA-mediated regulation of all1871.


Introduction
Small non-coding RNAs (sRNAs) are relevant players in regulatory circuits affecting essentially every aspect of bacterial physiology. These types of molecules are usually post-transcriptional regulators fine-tuning the responses to different environmental conditions [1]. In fact, the interplay between the regulation exerted by transcription factors and that exerted by small RNAs produces complex regulatory circuits in the form of feed-forward loops (coherent or incoherent) involving a transcription factor, an sRNA and their regulated target(s) [2,3].
Global nitrogen regulation is controlled in cyanobacteria by NtcA, a protein that belongs to the CRP/FNR family of transcriptional regulators [4]. Direct binding of NtcA to the corresponding promoters accounts for regulation of many nitrogen-regulated genes [5][6][7]. However, the mechanisms involved in expression of some nitrogen-regulated genes whose promoters do not contain NtcA binding sites might involve the participation of other NtcA-regulated factor(s). NtcA has been shown to regulate expression of several sRNAs, some of them with a wide distribution among phylogenetically distant cyanobacteria [5,8,9]. Among these sRNAs, NsiR4 (nitrogen-stress inducible RNA 4) is involved in nitrogen assimilation control via regulation of IF7, the inactivating factor of the key enzyme glutamine synthetase [8]. NsrR1 (nitrogen-stress repressed RNA 1) modulates translation of NblA [10], a protein Life 2020, 10, 54 2 of 16 required for the degradation of phycobilisomes that provide amino acids as a source of nitrogen under nitrogen deficiency [11]. Transcription of both gifA (encoding IF7) and nblA is directly regulated by NtcA [5,12], therefore the post-transcriptional regulation exerted on these transcripts by NsiR4 and NsrR1, respectively, constitutes a second level of NtcA-mediated, indirect regulation.
In the absence of combined nitrogen, filamentous cyanobacteria such as model strain Nostoc sp. PCC 7120 differentiate heterocysts, specialized cells devoted to fixation of atmospheric nitrogen [13,14]. Differentiation of functional heterocysts is ultimately under control of NtcA, but also requires HetR, a regulator specifically involved in cellular differentiation. The nitrogen-regulated, HetR-dependent transcriptome includes transcripts for genes involved in specific aspects of heterocyst physiology, such as the sequential deposition of specialized envelopes or the fixation of nitrogen by the enzyme nitrogenase. The HetR-dependent transcriptome also includes non-coding transcripts, both antisense and small RNAs, that would participate in the metabolic reprogramming that takes place in heterocysts [15,16], again pointing to the relevance of post-transcriptional regulation on cyanobacterial physiology.
In this work, we identify all1871 as a gene required for heterocyst function and describe its regulation by NsrR1. Expression of all1871 is induced upon nitrogen step down, but its induction does not require NtcA or HetR. We verify that NsrR1 regulates accumulation of All1871 at the post-transcriptional level by its interaction with the 5 -UTR of all1871.

Reporter Assays for In Vivo Verification of Targets
For the experimental target verification in E. coli, we used a previously described reporter system [18] and the superfolder green fluorescent protein (sfGFP) plasmid pXG10-SF [19]. The 5 -UTR of all1871, from the transcriptional start site (TSS) at position −137 with respect to the initiation codon (coordinate 2234072) to 60 nucleotides within the coding region, containing the predicted NsrR1 interaction sequence, was amplified from genomic DNA using oligonucleotides 247 and 248 (see Table S2 for oligonucleotide sequences and description). The information about the TSS was taken from [5]. The corresponding polymerase chain reaction (PCR) product was digested with NsiI and XbaI and cloned into the vector pXG10-SF digested with NsiI and NheI, resulting in plasmid pIAE9, bearing a translational fusion of a truncated All1871 protein with sfGFP (see Table S3 for plasmid descriptions). For NsrR1 expression in E. coli, plasmid pAVN1 [10] was used (Table S3).
Mutation U51G (Mut-51) was introduced in NsrR1 by overlapping PCR using primer pairs 197 and 296, and 198 and 295, and cloned as described for the wild type version [10] generating plasmid pIAE20 (Table S3). The compensatory mutation in the 5 -UTR of all1871 (Comp-51) was generated in the same way with primer pairs 247 and 304, and 303 and 248, and cloned as described above for the wild-type version resulting in plasmid pIAE22 (Table S3). The sequences of inserts in plasmids containing NsrR1 and all1871-sfgfp fusions are shown in Tables S4 and S5, respectively. Life 2020, 10, 54

of 16
For testing various combinations of both plasmids, these were introduced into E. coli DH5α. Plasmid pJV300 [20] was used as a control expressing an unrelated RNA. Plasmid pXG0 [18] was used as control for background fluorescence. Fluorescence measurements were done with a microplate reader (Varioskan) using liquid cultures from eight individual colonies bearing each combination of plasmids, and normalized to the OD 600 of each culture as described previously [21]. Fluorescence was also visualized in E. coli cells plated on solid LB medium by excitation with a 302-nm wavelength lamp.

RNA Isolation, Northern Blot and Primer Extension Analysis
RNA samples were isolated from cells collected at different times after removing combined nitrogen (ammonium) from the media. Alternatively, cells were grown in media lacking combined nitrogen and RNA was isolated from cells collected at different times after the addition of 10 mM NH 4 Cl and 20 mM TES buffer. Total RNA was isolated using hot phenol as described [22] with modifications [9]. Northern blot hybridization was performed as previously described [23,24]. Strand-specific 32 P-labelled probes for Northern blot were prepared with Taq DNA polymerase using a PCR fragment as template in a reaction with [α-32 P]dCTP and one single oligonucleotide as primer (corresponding to the complementary strand of the sRNA or mRNA to be detected). Hybridization to rnpB [25] was used as loading and transfer control. Hybridization signals were quantified on a Cyclone Storage Phosphor System with Optiquant software (PerkinElmer). Primer extension analysis of 5 ends of all1871 was carried out as previously described [23] using 5 µg of total RNA and oligonucleotide 161 labeled with [γ-32 P]ATP.

In Vitro Synthesis and Labelling of RNA
The DNA templates for the in vitro transcription of NsrR1 and all1871 5 -UTR RNA were generated by PCR with a forward primer that includes a T7 promoter sequence and three extra Gs upstream the 5 -end of the coded RNA, and a reverse primer corresponding to the 3 end of the RNA (see Tables S2 and S6). The all1871 5 -UTR fragment extends from the TSS at position −137 to 60 nucleotides downstream the translational start. RNA transcripts were generated with the MEGAscript High-Yield Transcription Kit (AM1333, Ambion). After transcription, RNAs were treated with DNase I and purified by phenol and chloroform extraction, ethanol-precipitated at -20 • C, and washed with 70% ethanol. In vitro transcribed RNAs were 5 -labelled and purified as described [10].

In Vitro Structure Probing and Footprinting
We mixed 0.1 pmol (about 50,000 cpm) of labeled NsrR1 RNA in 7 µL with 2 pmol of unlabelled all1871 5 -UTR RNA, denatured for 1 min at 95 • C and chilled on ice for 5 min, followed by the addition of 1 µL of 1 mg/mL yeast RNA (Ambion AM7118) and 1 µL of 10× structure buffer (Ambion). The samples were incubated further for 15 min at 37 • C. Treatment with RNase T1, RNase A or lead(II) acetate were performed as described [10].
An alkaline ladder was obtained by incubating 0.2 pmol of 5 -labelled RNA at 95 • C for 3 min in 7.5 µL of alkaline hydrolysis buffer (Ambion) containing 1.5 µg of yeast RNA (Ambion AM7118). Reactions were stopped by the addition of 15 µL of denaturing formamide loading buffer.
RNase T1 G ladders were obtained by incubating 0.1 pmol of 5 -labelled RNA and 1 µL of 1 mg/mL yeast RNA (Ambion AM7118) in 9 µL sequencing buffer (Ambion) for 10 min at 50 • C, followed by the addition of 1 µL of 0.1 U/mL RNase T1 (Ambion AM2283) and incubation at room temperature for 15 min. Reactions were stopped by the addition of 20 µL of Inactivation/Precipitation buffer (Ambion) and incubation at −20 • C for 15 min. The precipitate was washed with 70% ethanol and resuspended in 3-7 µL of denaturing formamide loading buffer.
All samples were run on 10% polyacrylamide, 7 M urea gels and bands visualized with a Cyclone Storage Phosphor System (PerkinElmer).

Expression and Purification of Protein All1871 and Western Blot
To produce His-tagged All1871 protein, the all1871 gene was amplified using Nostoc DNA as template and primers 335 and 336, and the PCR product was cloned in vector pET-28a (+) (Novagen) using NcoI and XhoI, producing plasmid pIAE30. Plasmid pIAE30, containing downstream a T7 polymerase-dependent promoter the all1871 gene fused at the 3 end to a sequence encoding a His 6 -tag, was transferred by electroporation to E. coli BL21-(DE3)-RIL, in which the gene encoding T7 RNA polymerase is under the control of an isopropyl-β-D-1-thiogalactopyranoside (IPTG)-regulated promoter. A 25-mL pre-inoculum of this strain was grown overnight in LB medium supplemented with chloramphenicol and kanamycin and used to inoculate 275 mL of the same medium. The culture was incubated at 37 • C until OD 600 = 0.6. Recombinant All1871 expression was induced by the addition of 1 mM IPTG. After 4 h at 37 • C, cells were collected and resuspended in 20 mM sodium phosphate buffer (pH 7.2) containing 6 M urea, 500 mM NaCl, 5 mM imidazole and 1 mM phenylmethylsulfonyl fluoride (6 mL/g of cells). Cells were broken by sonication and after centrifugation at 15,000× g (15 min, 4 • C), and the His 6 -All1871 protein was purified from the supernatant by chromatography through a 1-mL HisTrap HP column (GE Healthcare), using an imidazole gradient in the same buffer to elute the retained proteins. An additional purification step was carried out by size-exclusion chromatography in a HiLoad 16/60 Superdex 75 column (Pharmacia) using 20 mM sodium phosphate buffer (pH 7.2) containing 2 M urea and 150 mM NaCl. A total amount of 3.5 mg of purified protein was used in seven subcutaneous injections of a rabbit to produce antibodies in the Animal Production and Experimentation Center, Universidad de Sevilla (Seville, Spain). Antiserum was recovered several times up to five months after the first injection and stored at −80 • C until used. Antibodies specific for All1871 were purified from the serum by affinity chromatography on immobilized His-tagged All1871 using the AminoLink ® Plus Immobilization Kit (ThermoFisher, Waltham, MA, USA) following the manufacturer's instructions.
For Western blot analysis, E. coli cells were resuspended in sodium dodecyl sulphatepolyacrylamide gel electrophoresis (SDS-PAGE) loading buffer and the proteins fractionated on 15% SDS-PAGE. Antibodies against All1871 (see above), GFP (Roche) and E. coli GroEL (SIGMA-ALDRICH) were used. The ECL Plus immunoblotting system (GE Healthcare) was used to detect the different antibodies using anti-rabbit (SIGMA-ALDRICH) or anti-mouse (Bio-Rad) horse radish peroxidase conjugated secondary antibodies.
For Western blot analysis of Nostoc proteins, soluble fractions of cell-free extracts were used. Cells from 25 mL of culture were resuspended in 500 µL of Tris-HCl buffer (pH 8) containing 2 mM β-mercaptoethanol and protease inhibitor cocktail (cOmplete TM ultra tablets, EDTA-free, Roche) in an Eppendorf 1.5 mL tube. A volume of approximately 75 µL of glass beads (0.25-0.3 mm diameter) was added and the suspension subjected to seven cycles of 1 min vortex followed by 1 min on ice. The resulting extract was centrifuged 3 min at 3000× g at 4 • C and the supernatant further centrifuged for 30 min at 16,000× g at 4 • C. The supernatant of the last centrifugation constitutes the soluble fraction.

Generation and Complementation of all1871 Mutant Strain
To generate a strain lacking all1871, two overlapping fragments were amplified by PCR using as template genomic DNA with oligonucleotides 162 and 163 and oligonucleotides 164 and 165, respectively (Table S2). The resulting products were then used as templates for a third PCR with oligonucleotides 162 and 165 resulting in deletion of sequences corresponding to all1871 and the generation of a unique XhoI site between the two amplified fragments. The fragment was cloned into pSpark (Canvax Biotech), rendering pSAM318 and its sequence was verified (Eurofins Genomics). After digestion with BamHI at the sites provided by oligonucleotides 162 and 165, the fragment was cloned into BamHI-digested sacB-containing Sm R Sp R vector pCSRO [26], rendering pSAM324. A Nm R gene was excised from pRL278 [27] as a SalI-XhoI fragment and cloned into the XhoI site in pSAM324, rendering pSAM326, which was transferred to Nostoc sp. strain PCC 7120 by conjugation [28], with selection for resistance to Nm. Cultures of the exconjugants obtained were used to select for Life 2020, 10, 54 5 of 16 clones resistant to 5% sucrose [29], and individual sucrose resistant colonies were checked by PCR using flanking oligonucleotides 162 and 180. Clones bearing the all1871 region interrupted by the Nm R gene were named all1871::Nm.
Plasmid pIAE65 was constructed to express all1871 from the trc promoter. A PCR fragment was amplified using oligonucleotides 247 and 735, digested with NsiI and XhoI and cloned into NsiI and XhoI-digested pMBA37 [15]. pIAE65 was transferred to strain all1871::Nm by conjugation with selection of Sm R Sp R cells.

Construction of a Strain Bearing a Translational Fusion all1871-gfpmut2
In order to analyze spatial expression of all1871 along Nostoc filaments, plasmid pELV75 (Table S3) was constructed. A fragment containing the sequence from position −200 with respect to the TSS plus the entire all1871 gene was amplified using oligonucleotides 451 and 596. The fragment was digested with ClaI and EcoRV and cloned in pCSAM147 [30], in frame with the gpfmut2 gene, rendering pELV73. The EcoRI fragment from pELV73 containing the all1871-gfpmut2 translational fusion was cloned into pCSV3 [31] producing pELV75, that was transferred to Nostoc by conjugation with selection for Sm R Sp R cells.

Microscopy
Fluorescence of Nostoc sp. PCC 7120 filaments carrying plasmid pELV75 growing on top of solidified nitrogen-free medium, was analyzed by confocal microscopy and quantified as described [32] using a Leica HCX PLAN-APO 63× 1.4 NA oil immersion objective attached to a Leica TCS SP2 laser-scanning confocal microscope. Samples were excited at 488 nm by an argon ion laser and the fluorescent emission was monitored by collection across windows of 500-538 nm (GFP) and 630-700 nm (cyanobacterial autofluorescence). Filaments were stained with Alcian blue and visualized at the microscope as described [33].

NsrR1 Interacts with all1871 mRNA 5 -UTR
NsrR1 (nitrogen stress-repressed RNA 1) is a small RNA previously described to post-transcriptionally regulate expression of nblA [10]. A prediction of mRNAs possibly regulated by NsrR1 also identified the 5 -UTR of gene all1871 as likely interacting with NsrR1 [10]. all1871 would encode a conserved protein of unknown function. The high score of the prediction (p-value = 6.11 × 10 −9 ), together with the conservation of predicted interactions between NsrR1 homologs and all1871 homologs in most cyanobacteria encoding NsrR1 ( Figure S1), prompted us to further analyze a possible post-transcriptional regulation of all1871 by NsrR1. Figure 1 shows the predicted interaction between NsrR1 from Nostoc sp. PCC 7120 and the 5 -UTR of all1871, that extends from position −40 to −3, overlaps the translation initiation region and therefore is expected to affect translation of the mRNA ( Figure 1A). To verify the interaction between NsrR1 and the mRNA of all1871, we used a heterologous reporter assay in E. coli [18], in which the 5 -UTR of all1871, from the TSS at position −137 with respect to the initiation codon (Mitschke et al., 2011), plus 60 nucleotides of the coding sequence of all1871 were fused to the sfgfp gene and co-expressed in E. coli with NsrR1 or with a control RNA. Accumulation of the GFP protein was measured in E. coli cells bearing different combinations of plasmids. The translation initiation region of all1871 was functional in E. coli resulting in significant GFP protein accumulation and GFP fluorescence ( Figure 1B-D). The GFP fluorescence of cells bearing the all1871::sfgfp fusion (and the amount of GFP protein) decreased to less than 20% of the control when NsrR1 was co-expressed, indicating a direct interaction between NsrR1 and the 5 -UTR of all1871, which affects translation ( Figure 1B-C).
To verify the interaction at the predicted site, a point mutation was introduced in NsrR1 affecting the predicted helix between NsrR1 and the 5 -UTR of all1871 (nucleotide 51, U to G). A compensatory Life 2020, 10, 54 6 of 16 mutation was introduced in the corresponding positions of the 5 -UTR of all1871 (nucleotide −13 with respect to the start codon, G to C) and different combinations of the wild-type and mutated versions of both NsrR1 and 5 -UTR of all1871 were co-expressed. Mutation of nucleotide 51 in NsrR1 reduced the interaction between NsrR1 and the 5 -UTR of all1871, as suggested by the lower degree of fluorescence reduction with respect to the control ( Figure 1C). When the mutated version of NsrR1 was combined with the mutated version of the mRNA containing the compensatory change, base pairing was restored resulting in a stronger fluorescence reduction than with wild-type NsrR1. Because of the long region of interaction, mutation of one single nucleotide produces a small effect, but differences observed are statistically significant. These data support a direct interaction of NsrR1 with the 5 -UTR of the all1871 mRNA that affects translation of All1871.
Life 2020, 10, x FOR PEER REVIEW 6 of 16 of both NsrR1 and 5'-UTR of all1871 were co-expressed. Mutation of nucleotide 51 in NsrR1 reduced the interaction between NsrR1 and the 5'-UTR of all1871, as suggested by the lower degree of fluorescence reduction with respect to the control ( Figure 1C). When the mutated version of NsrR1 was combined with the mutated version of the mRNA containing the compensatory change, base pairing was restored resulting in a stronger fluorescence reduction than with wild-type NsrR1.
Because of the long region of interaction, mutation of one single nucleotide produces a small effect, but differences observed are statistically significant. These data support a direct interaction of NsrR1 with the 5'-UTR of the all1871 mRNA that affects translation of All1871. In addition, we have studied the interaction between NsrR1 and the all1871 mRNA by in vitro footprinting analysis. 32 P-labelled NsrR1 was incubated with unlabeled all1871 mRNA (a fragment extending from positions −137 to +60 with respect to the start of the coding sequence) and probed with RNase T1, RNase A or lead(II) acetate (Figure 2A). A clear footprint was detected between positions 45 and 62 of NsrR1 (highlighted in the secondary structure model, Figure 2B), in good agreement with the bioinformatic prediction ( Figure 1A). These in vitro results, therefore, confirm the in vivo results obtained from the analysis in E. coli using the sfGFP fusion system. with the control plasmid pJV300. Strains growing on LB agar plates were photographed under ultraviolet (UV) light.
In addition, we have studied the interaction between NsrR1 and the all1871 mRNA by in vitro footprinting analysis. 32 P-labelled NsrR1 was incubated with unlabeled all1871 mRNA (a fragment extending from positions −137 to +60 with respect to the start of the coding sequence) and probed with RNase T1, RNase A or lead(II) acetate (Figure 2A). A clear footprint was detected between positions 45 and 62 of NsrR1 (highlighted in the secondary structure model, Figure 2B), in good agreement with the bioinformatic prediction ( Figure 1A). These in vitro results, therefore, confirm the in vivo results obtained from the analysis in E. coli using the sfGFP fusion system.

Expression of all1871 Is Regulated by Nitrogen Availability But Does Not Require NtcA or HetR
According to global transcriptomic analyses, accumulation of the all1871 transcript is induced in response to nitrogen deficiency [16,35]. We have analyzed transcription from the TSS identified at position 2234072r [5] in the wild type strain and in two mutant derivatives, ntcA mutant strain CSE2 [36] and hetR mutant strain DR884a [27]. Although in all strains there is significant expression in the presence of ammonium, transcription is similarly induced upon nitrogen step down in the wild type and in both mutant strains, suggesting the induction of transcription does not require NtcA or HetR (Figure 3). According to primer extension analysis, induction is transient both in the wild type and the ntcA strain, with a decreased amount of transcript at 24 h vs. 8 h after nitrogen removal, but in the hetR mutant the accumulation of transcript continues to be strong even at 24 h after nitrogen removal.
RNase A or Pb(II) footprinting of the interaction between NsrR1 and the 5'-UTR of all1871. -/+ above lanes indicate absence/presence of all1871 5' UTR. The protected area is indicated by a vertical bar. NsrR1 was 5' end-labelled. C, untreated control; OH, alkaline ladder; T1, RNase T1 ladder. Nucleotide positions of NsrR1 are shown on the left. (B) The nucleotides in NsrR1 involved in the interaction with the all1871 5'-UTR are indicated in grey on the previously described secondary structure model of NsrR1 [10]. The nucleotide changed (U to G) in version Mut-51 of NsrR1 is also indicated.

Expression of all1871 is Regulated by Nitrogen Availability but Does Not Require NtcA or HetR
According to global transcriptomic analyses, accumulation of the all1871 transcript is induced in response to nitrogen deficiency [16,35]. We have analyzed transcription from the TSS identified at position 2234072r [5] in the wild type strain and in two mutant derivatives, ntcA mutant strain CSE2 [36] and hetR mutant strain DR884a [27]. Although in all strains there is significant expression in the presence of ammonium, transcription is similarly induced upon nitrogen step down in the wild type and in both mutant strains, suggesting the induction of transcription does not require NtcA or HetR (Figure 3). According to primer extension analysis, induction is transient both in the wild type and the ntcA strain, with a decreased amount of transcript at 24 hours vs. 8 hours after nitrogen removal, but in the hetR mutant the accumulation of transcript continues to be strong even at 24 hours after nitrogen removal.

Expression of all1871 is Reduced by NsrR1 in Nostoc sp. PCC 7120
In order to study in vivo in Nostoc the possible effect of NsrR1 on all1871 expression, we used a mutant strain that lacks NsrR1 (∆nsrR1) [10]. Cells of strain ∆nsrR1 have no apparent difference in growth with respect to wild-type cells in media containing nitrate, ammonia, or lacking a source of combined nitrogen [10].
We have previously shown that transcription of NsrR1 is only partially (and transiently) repressed upon nitrogen step down, whereas maximal expression of NsrR1 is achieved upon ammonium addition to cells growing in the absence of combined nitrogen [10]. Therefore, to maximize the difference between wild-type cells expressing NsrR1 and cells lacking NsrR1 (∆nsrR1 strain), we chose to analyze cells grown in the absence of combined nitrogen (N2) and compared them with cells grown in the absence of combined nitrogen to which ammonium was added and incubation continued for 4, 8 or 24 hours (Figure 4).

Expression of all1871 Is Reduced by NsrR1 in Nostoc sp. PCC 7120
In order to study in vivo in Nostoc the possible effect of NsrR1 on all1871 expression, we used a mutant strain that lacks NsrR1 (∆nsrR1) [10]. Cells of strain ∆nsrR1 have no apparent difference in growth with respect to wild-type cells in media containing nitrate, ammonia, or lacking a source of combined nitrogen [10].
We have previously shown that transcription of NsrR1 is only partially (and transiently) repressed upon nitrogen step down, whereas maximal expression of NsrR1 is achieved upon ammonium addition to cells growing in the absence of combined nitrogen [10]. Therefore, to maximize the difference between wild-type cells expressing NsrR1 and cells lacking NsrR1 (∆nsrR1 strain), we chose to analyze cells grown in the absence of combined nitrogen (N 2 ) and compared them with cells grown in the absence of combined nitrogen to which ammonium was added and incubation continued for 4, 8 or 24 h (Figure 4).
As previously described, expression of NsrR1 was induced and reached its highest level 8 h after ammonium addition ( Figure 4A, middle panel). Concomitantly, expression of all1871 was repressed in the wild type, with a minimum in the sample corresponding to 8 h after ammonium addition ( Figure 4A, upper panel). Furthermore, repression of all1871 was weaker in the strain lacking NsrR1, suggesting NsrR1 has a significant negative effect on the accumulation of all1871 mRNA in Nostoc, consistent with the interaction observed in the E. coli assay between NsrR1 and the all1871 mRNA. The effects of NsrR1 on the accumulation of All1871 protein were also analyzed by Western blot using antibodies we have generated against purified recombinant All1871 protein. The levels of All1871 Life 2020, 10, 54 9 of 16 protein observed in extracts of strain ∆nsrR1 were about five-fold higher than those in the wild-type strain in the absence of combined nitrogen ( Figure 4B). Consistent with the mRNA levels ( Figure 4A), the reduction of the amount of All1871 protein in response to ammonium addition was stronger in the wild-type strain than in the ∆nsrR1 strain lacking NsrR1. Lower panels show detection of GroEL, used as loading and transfer control. Quantification of All1871 protein is shown at the bottom, relative to the amount present in the wild type strain in N2. Quantification was performed with ImageLab software (Bio-Rad) using the amount of GroEL for normalization. One representative experiment is shown.
As previously described, expression of NsrR1 was induced and reached its highest level 8 hours after ammonium addition ( Figure 4A, middle panel). Concomitantly, expression of all1871 was repressed in the wild type, with a minimum in the sample corresponding to 8 hours after ammonium addition ( Figure 4A, upper panel). Furthermore, repression of all1871 was weaker in the strain lacking NsrR1, suggesting NsrR1 has a significant negative effect on the accumulation of all1871 mRNA in Nostoc, consistent with the interaction observed in the E. coli assay between NsrR1 and the all1871 mRNA. The effects of NsrR1 on the accumulation of All1871 protein were also analyzed by Western blot using antibodies we have generated against purified recombinant All1871 protein. The levels of All1871 protein observed in extracts of strain ∆nsrR1 were about five-fold higher than those Cells were grown in nitrogen-free liquid medium for one week (steady-state N 2 -fixing cultures) and 10 mM ammonium chloride was added to increase expression of NsrR1. Samples were taken before ammonium addition (0) and at the number of hours indicated after ammonium addition. The upper panel shows hybridization to the all1871 probe. The middle panel shows hybridization to the probe for NsrR1. The lower panel shows hybridization to a probe for rnpB used as loading and transfer control. Quantification of all1871 mRNA accumulation is indicated on the right, relative to the amount present in the wild type strain in N 2 using the amount of rnpB for normalization. Results from two technical replicates were averaged. T-test p-value < 0.05*; < 0.01**. (B) Accumulation of the All1871 protein was determined by Western blot in samples containing 40 µg of soluble fraction from cells analyzed in (A). Upper panels show detection of All1871. Lower panels show detection of GroEL, used as loading and transfer control. Quantification of All1871 protein is shown at the bottom, relative to the amount present in the wild type strain in N 2 . Quantification was performed with ImageLab software (Bio-Rad) using the amount of GroEL for normalization. One representative experiment is shown.

All1871 Is Differentially Expressed in Heterocysts and Required for Diazotrophic Growth But Not for Heterocyst Differentiation
In order to obtain an insight into a possible function of All1871, we analyzed expression of all1871 along filaments of Nostoc. We prepared plasmid pELV75 containing a segment from position −200 Life 2020, 10, 54 10 of 16 with respect to the TSS of all1871 plus a translational fusion between the entire all1871 gene and the gfpmut2 gene. This plasmid was introduced in Nostoc by conjugation. Integration of the plasmid in the region encoding all1871 leads to a partial duplication of this region in which the translational fusion is preceded by the natural context of all1871 in the wild type strain ( Figure 5A).
In order to obtain an insight into a possible function of All1871, we analyzed expression of all1871 along filaments of Nostoc. We prepared plasmid pELV75 containing a segment from position −200 with respect to the TSS of all1871 plus a translational fusion between the entire all1871 gene and the gfpmut2 gene. This plasmid was introduced in Nostoc by conjugation. Integration of the plasmid in the region encoding all1871 leads to a partial duplication of this region in which the translational fusion is preceded by the natural context of all1871 in the wild type strain ( Figure 5A). GFP fluorescence was analyzed by confocal fluorescence microscopy of filaments growing on top of medium lacking combined nitrogen ( Figure 5B). Quantification of the green signal (GPF) and the red autofluorescence along the filaments showed that although all cells showed green fluorescence, fluorescence peaks were associated with cells that had differentiated as heterocysts, as indicated by their larger size and reduced red autofluorescence.  GFP fluorescence was analyzed by confocal fluorescence microscopy of filaments growing on top of medium lacking combined nitrogen ( Figure 5B). Quantification of the green signal (GPF) and the red autofluorescence along the filaments showed that although all cells showed green fluorescence, fluorescence peaks were associated with cells that had differentiated as heterocysts, as indicated by their larger size and reduced red autofluorescence.
We then prepared an all1871 null mutant by interrupting the all1871 gene with a Nm R gene ( Figure S2A). Complete segregation of mutant chromosomes was verified by PCR amplification and Western blot with antibodies against All1871 ( Figure S2B-C). Cells of the all1871::Nm mutant were unable to grow on plates in the absence of combined nitrogen but showed no growth defect in the presence of combined nitrogen (nitrate) ( Figure 6A). The mutation was complemented by introduction of a plasmid bearing all1871 expressed from the trc promoter from E. coli, that provides constitutive Life 2020, 10, 54 11 of 16 expression in Nostoc (see e.g., [37]), leading to partial recovery of the wild-type phenotype, as indicated by the greenish growth of complemented cells vs. the blue-green growth of wild-type cells ( Figure 6A).
We then prepared an all1871 null mutant by interrupting the all1871 gene with a Nm R gene ( Figure S2A). Complete segregation of mutant chromosomes was verified by PCR amplification and Western blot with antibodies against All1871 ( Figure S2B-C). Cells of the all1871::Nm mutant were unable to grow on plates in the absence of combined nitrogen but showed no growth defect in the presence of combined nitrogen (nitrate) ( Figure 6A). The mutation was complemented by introduction of a plasmid bearing all1871 expressed from the trc promoter from E. coli, that provides constitutive expression in Nostoc (see e.g., [37]), leading to partial recovery of the wild-type phenotype, as indicated by the greenish growth of complemented cells vs. the blue-green growth of wild-type cells ( Figure 6A).  We wondered whether the observed defects in diazotrophic growth of the all1871::Nm strain were due to lack of heterocyst differentiation or heterocyst function. Because steady-state nitrogen-fixing cultures could not be obtained, we analyzed morphological differentiation of heterocysts 26 h after combined nitrogen removal by staining the cells with Alcian blue, a molecule that binds to the external polysaccharide layer of heterocysts. Figure 6B shows the presence of Alcian blue-stained cells in a regular pattern similar to the wild type in the mutant strain all1871::Nm as well as in the complemented strain all1871::Nm + P trc -all1871, indicating differentiation of heterocysts took place in both strains, at least to the relatively initial stage in which polysaccharides are deposited outside the outer membrane of the vegetative cells undergoing differentiation into heterocysts. As an indication of heterocyst maturity, transcription of the nifHDK genes encoding nitrogenase was also analyzed by Northern blot ( Figure 6C). Transcription of nifHDK seems delayed and reduced in the all1871::Nm mutant. Again, complementation with a plasmid bearing all1871 expressed from the trc promoter from E. coli leads to recovery of the wild-type timing of nifHDK expression.
Finally, we have measured the nitrogenase activity of the different strains (Table 1). Strain all1871::Nm had no detectable nitrogenase activity in either oxic or anoxic conditions. This result excludes the possibility that the absence of activity in strain all1871::Nm was due to a defect in the maturation of heterocyst envelopes that results in the presence of inactivating O 2 amounts inside the heterocyst.

Discussion
Small RNAs are important components in regulatory networks that involve classical transcription factors. For instance, in E. coli, members of the CRP/FNR family of transcriptional regulators are known to control the expression of several small RNAs, all of them contributing to the regulatory effects exerted by these two major transcription factors [2]. Cyanobacterial transcription factors also regulate the expression of small RNAs that exert regulatory functions involved in the adaptation to different environmental situations [38]. For instance, the transcriptional regulator RpaB and small RNA PsrR1 constitute a feed-forward loop controlling acclimation to different light intensities [39]. Accumulating evidence concerning nitrogen-regulated non-coding RNAs indicates this type of molecules is involved in NtcA-mediated post-transcriptional regulation [5,9]. One case analyzed in detail is NsiR4, whose transcription is induced in response to nitrogen deficiency and is involved in post-transcriptional regulation of glutamine synthetase [8]. Expression of another sRNA, NsrR1, is repressed by NtcA in response to nitrogen deficiency. Among predicted targets of NsrR1 are nblA [10], and all1871, whose interaction with NsrR1 we describe in this work.
Interaction between homologs of NsrR1 and homologs of all1871 is conserved in many cyanobacteria encoding NsrR1 ( Figure S1), consistent with the observation that all1871 has the highest score in CopraRNA predictions of candidates to be regulated by NsrR1 [10]. Using an in vivo reporter system established in E. coli we demonstrate that NsrR1 represses translation, leading to reduced expression of an all1871::sfGFP fusion in the presence of NsrR1. A direct interaction of NsrR1 with the predicted region in the 5 -UTR of all1871 was also verified by a point mutation in the region involved ( Figure 1) and by in vitro footprinting experiments (Figure 2).
We have demonstrated higher accumulation of all1871 transcripts in a strain of Nostoc sp. PCC 7120 that lacks NsrR1 (∆nsrR1) than in wild-type cells cultured in the absence of combined nitrogen ( Figure 4A). Upon addition of ammonium, that induces NsrR1 expression, the amount of all1871 mRNA is strongly reduced in the wild type but not in the ∆nsrR1 strain. Similarly, the amount of All1871 protein, detected with a specific antibody ( Figure 4B), is much higher in cells lacking NsrR1 than in the wild-type strain. Upon the addition of ammonium, the amount of All1871 protein is reduced in the wild type, becoming barely detectable 8 h after ammonium addition, but only slightly reduced in the ∆nsrR1 strain. The magnitude of the repression upon ammonium addition is higher at the protein level than at the transcript level, in agreement with NsrR1 inhibiting translation through its interaction with the 5 -UTR of all1871. The reduced accumulation of all1871 mRNA in the presence of NsrR1 can be attributed to destabilization of the mRNA when translation is inhibited.
Upregulation of all1871 upon nitrogen deprivation would be mediated, at least in part, by alleviating the repression exerted by NsrR1, whose transcription is repressed upon nitrogen stress. NsrR1 repression is only partially dependent on NtcA, with an additional factor possibly involved [10], explaining that up-regulation of all1871 is independent of NtcA ( Figure 3). The observation that there is partial repression of all1871 expression upon ammonium addition even in the ∆nsrR1 mutant strain ( Figure 4) points to some additional factor(s) being involved in regulation of all1871. In any case, because the interaction between NsrR1 and all1871 takes place at the translation initiation region, even if another regulatory mechanism influences transcription of all1871, the accumulation of the All1871 protein would be ultimately regulated by nitrogen availability through NsrR1 (Figure 7). Life 2020, 10, x FOR PEER REVIEW 13 of 16 score in CopraRNA predictions of candidates to be regulated by NsrR1 [10]. Using an in vivo reporter system established in E. coli we demonstrate that NsrR1 represses translation, leading to reduced expression of an all1871::sfGFP fusion in the presence of NsrR1. A direct interaction of NsrR1 with the predicted region in the 5'-UTR of all1871 was also verified by a point mutation in the region involved ( Figure 1) and by in vitro footprinting experiments ( Figure 2). We have demonstrated higher accumulation of all1871 transcripts in a strain of Nostoc sp. PCC 7120 that lacks NsrR1 (∆nsrR1) than in wild-type cells cultured in the absence of combined nitrogen ( Figure 4A). Upon addition of ammonium, that induces NsrR1 expression, the amount of all1871 mRNA is strongly reduced in the wild type but not in the ∆nsrR1 strain. Similarly, the amount of All1871 protein, detected with a specific antibody ( Figure 4B), is much higher in cells lacking NsrR1 than in the wild-type strain. Upon the addition of ammonium, the amount of All1871 protein is reduced in the wild type, becoming barely detectable 8 h after ammonium addition, but only slightly reduced in the ∆nsrR1 strain. The magnitude of the repression upon ammonium addition is higher at the protein level than at the transcript level, in agreement with NsrR1 inhibiting translation through its interaction with the 5'-UTR of all1871. The reduced accumulation of all1871 mRNA in the presence of NsrR1 can be attributed to destabilization of the mRNA when translation is inhibited.
Upregulation of all1871 upon nitrogen deprivation would be mediated, at least in part, by alleviating the repression exerted by NsrR1, whose transcription is repressed upon nitrogen stress. NsrR1 repression is only partially dependent on NtcA, with an additional factor possibly involved [10], explaining that upregulation of all1871 is independent of NtcA ( Figure 3). The observation that there is partial repression of all1871 expression upon ammonium addition even in the ∆nsrR1 mutant strain (Figure 4) points to some additional factor(s) being involved in regulation of all1871. In any case, because the interaction between NsrR1 and all1871 takes place at the translation initiation region, even if another regulatory mechanism influences transcription of all1871, the accumulation of the All1871 protein would be ultimately regulated by nitrogen availability through NsrR1 (Figure 7). By means of a translational fusion to the gfp gene we have shown higher expression of all1871 in heterocysts than in vegetative cells ( Figure 5). We have also shown that expression of all1871 does not require HetR (Figure 3) suggesting it is not heterocyst-specific. Taken together these observations point to the operation of post-transcriptional regulatory mechanisms leading to differential accumulation of all1871 transcripts in heterocysts vs. vegetative cells. Because NtcA is subjected to differential accumulation in heterocysts [31,40], the amount of NsrR1 is also expected to be different. A higher amount of NtcA in heterocysts would result in lower levels of NsrR1, and consequently increased translation of All1871 in these specialized cells. NsrR1 would thus contribute to differential expression in heterocysts of a transcript found to be required for diazotrophic growth. Whether the By means of a translational fusion to the gfp gene we have shown higher expression of all1871 in heterocysts than in vegetative cells ( Figure 5). We have also shown that expression of all1871 does not require HetR (Figure 3) suggesting it is not heterocyst-specific. Taken together these observations point to the operation of post-transcriptional regulatory mechanisms leading to differential accumulation of all1871 transcripts in heterocysts vs. vegetative cells. Because NtcA is subjected to differential accumulation in heterocysts [31,40], the amount of NsrR1 is also expected to be different. A higher amount of NtcA in heterocysts would result in lower levels of NsrR1, and consequently increased translation of All1871 in these specialized cells. NsrR1 would thus contribute to differential expression in heterocysts of a transcript found to be required for diazotrophic growth. Whether the stronger accumulation of All1871-GFP protein in heterocysts ( Figure 5) is only a consequence of reduced post-transcriptional regulation by NsrR1 or involves additional differential regulation in these specialized cells, is currently unknown.
All1871 is conserved in many unicellular and filamentous strains of cyanobacteria. Its function is unknown and it does not contain domains of known function. Strong accumulation in heterocysts together with the observation that its expression is regulated by NsrR1, a nitrogen-repressed sRNA, suggest a possible role in the response to nitrogen stress and/or in heterocyst function. A mutant lacking All1871 (all1871::Nm) could not grow in media lacking combined nitrogen ( Figure 6A). The inability to grow in media lacking combined nitrogen of the all1871::Nm strain is only partially complemented by constitutive expression of all1871 from P trc ( Figure 6A). Such phenotype could be explained by toxicity due to unregulated expression of all1871 in this strain, in contrast to the transient induction observed in the wild type upon nitrogen step-down. It can also be speculated that constitutive expression of all1871 in the complemented strain could lead to sequestration of NsrR1 affecting accumulation of other mRNAs also regulated by NsrR1, such as nblA [10]. However, it has been previously shown that cells lacking NsrR1 have no apparent phenotypic differences with respect to wild-type cells [10]. Despite its ability to develop Alcian blue-stained cells ( Figure 6B), suggestive of heterocyst differentiation, and the induction of nitrogenase transcription ( Figure 6C), all1871::Nm filaments lacked nitrogenase activity ( Table 1), indicating that All1871 is required for heterocyst function through currently unknown mechanisms. We have observed that in the absence of combined nitrogen, filaments of the all1871::Nm strain are extensively fragmented in liquid media (not shown). Because exchange of metabolites between vegetative cells and heterocysts is a requisite for sustained nitrogen fixation, filament fragmentation would preclude diazotrophic growth of the all1871::Nm strain.
In summary, in this work we describe a new gene required for diazotrophic growth, although not for heterocyst differentiation, and characterize its regulation by NsrR1, a nitrogen-regulated small RNA involved in acclimation to nitrogen deficiency.