Expanding the FurC (PerR) regulon in Anabaena (Nostoc) sp. PCC 7120: Genome-wide identification of novel direct targets uncovers FurC participation in central carbon metabolism regulation

Cristina Sarasa-Buisan; Jorge Guío; M. Luisa Peleato; María F. Fillat; Emma Sevilla

doi:10.1371/journal.pone.0289761

Abstract

FurC (PerR, Peroxide Response Regulator) from Anabaena sp. PCC 7120 (also known as Nostoc sp. PCC 7120) is a master regulator engaged in the modulation of relevant processes including the response to oxidative stress, photosynthesis and nitrogen fixation. Previous differential gene expression analysis of a furC-overexpressing strain (EB2770FurC) allowed the inference of a putative FurC DNA-binding consensus sequence. In the present work, more data concerning the regulon of the FurC protein were obtained through the searching of the putative FurC-box in the whole Anabaena sp. PCC 7120 genome. The total amount of novel FurC-DNA binding sites found in the promoter regions of genes with known function was validated by electrophoretic mobility shift assays (EMSA) identifying 22 new FurC targets. Some of these identified targets display relevant roles in nitrogen fixation (hetR and hgdC) and carbon assimilation processes (cmpR, glgP1 and opcA), suggesting that FurC could be an additional player for the harmonization of carbon and nitrogen metabolisms. Moreover, differential gene expression of a selection of newly identified FurC targets was measured by Real Time RT-PCR in the furC-overexpressing strain (EB2770FurC) comparing to Anabaena sp. PCC 7120 revealing that in most of these cases FurC could act as a transcriptional activator.

Citation: Sarasa-Buisan C, Guío J, Peleato ML, Fillat MF, Sevilla E (2023) Expanding the FurC (PerR) regulon in Anabaena (Nostoc) sp. PCC 7120: Genome-wide identification of novel direct targets uncovers FurC participation in central carbon metabolism regulation. PLoS ONE 18(8): e0289761. https://doi.org/10.1371/journal.pone.0289761

Editor: Muhammad Afzal, BOKU: Universitat fur Bodenkultur Wien, AUSTRIA

Received: April 21, 2023; Accepted: July 25, 2023; Published: August 7, 2023

Copyright: © 2023 Sarasa-Buisan et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Data Availability: All relevant data are within the paper and its Supporting Information files.

Funding: This work was founded by Ministerio de Ciencia, Innovación y Universidadaes (grant 438PID2019-104889GB-I00) and from Gobierno de Aragón (grant E35_20R Biología Estructural) The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests: The authors have declared that no competing interests exist.

Introduction

FUR (Ferric Uptake Regulator) proteins are prokaryotic metalloregulators mainly involved in the control of metal homeostasis and the concerted response to oxidative stress [1]. The Anabaena sp. PCC 7120 genome holds three paralogs belonging to the FUR family that are called FurA, FurB and FurC. FurC was firstly identified as a member of the family because it showed the distinctive features of FUR proteins [2] and it was later proposed that FurC was the PerR paralog in Anabaena sp. PCC 7120 [3]. This hypothesis was based on the fact that FurC was able to bind the regions upstream from genes encoding key proteins involved in the response to oxidative stress such as PrxA (Peroxiredoxin A) and SrxA (Sulfiredoxin A) and that displayed in vitro the canonical derepression mechanism of PerR proteins which is based on the metal catalyzed oxidation (MCO) process. In this process, FurC was inactivated by oxidation with Fe²⁺ under aerobic conditions hampering its DNA binding activity. Such inactivation can be prevented by previous incubation with excess of manganese. Later on it was demonstrated that the MCO performed by FurC happened in vivo in Anabaena sp. PCC 7120 cells allowing the derepression of antioxidant enzymes to face oxidative stress situations [4]. More recently, it was shown that in fact the irreversible inactivation of FurC/PerR by Fe²⁺ in aerobic conditions goes along with the incorporation of an oxygen atom into its structure as its observed for other PerR orthologs [5, 6]. This study also disclosed the FurC/PerR metal binding and oligomerization properties describing an additional layer of regulation of the protein based on cysteine-based reversible oxidation, increasing the complexity of its regulatory mechanism. Apart from its role in regulation of oxidative stress response genes, it was reported that FurC encompassed the regulation of other crucial processes in Anabaena sp. PCC 7120 such as the maintenance of the photosynthetic metabolism [4]. The overexpression of FurC in Anabaena sp. PCC 7120 led to changes in the composition and efficiency of the photosynthetic machinery and novel FurC targets were described such as the major thylakoid membrane protease FtsH which degrades D1 proteins in photoinhibition processes involved in PSII recycling [4].

Subsequently, differential gene expression analyses were carried out comparing the furC-overexpressing strain (EB2770FurC) transcriptional profile with that of the wild type strain (Anabaena sp. PCC 7120) under standard and diazotrophic growth conditions. These analyses evidenced that FurC was a global regulator involved in the control of a wide variety of processes including photosynthesis, iron homeostasis, energy metabolism, heterocyst development and nitrogen fixation [7]. In this study, several genes related to nitrogen metabolism were found to be directly regulated by FurC such as hetZ, asr1734, hepC, alr4973-75, nifH2, nifHDK, xisHI and rbrA [7]. Interestingly, the furC-overexpressing strain showed an impairment of heterocyst differentiation under combined nitrogen deficient conditions and serious difficulties to grow under diazotrophic conditions [7]. In addition, it was observed that FurC could also act as transcriptional activator displaying a dual role in its transcriptional regulation. The vast amount of novel direct targets of FurC retrieved from the differential gene expression analyses allowed the inference of a putative FurC-box yielding the FurC DNA-binding consensus sequence 5´- CAAAATCATAACGACTTTG-3´ [7].

Our previous studies suggested that FurC could be acting as a global player in Anabaena sp. PCC 7120. In the present work, we try to expand the direct regulatory network exerted by FurC and decipher new metabolic pathways modulated by this protein. To go further in the knowledge of the FurC regulon, an in silico identification of novel FurC-DNA binding sites was performed scanning the total Anabaena sp. PCC 7120 genome sequence with a more restrictive version of the putative FurC-box defined previously [7]. Furthermore, the potential FurC-regulated genes were analyzed by EMSA to identify actual direct targets of FurC. Additionally, Real Time RT-PCR experiments were performed comparing the transcriptional levels of the potential FurC targets in the furC overexpressing strain EB2770FurC and the wild-type Anabaena to study the potential effect of the FurC regulation. Among others, this study unveiled several FurC direct targets displaying important roles in nitrogen and carbon metabolisms.

Materials and methods

Bioinformatic prediction of FurC putative binding sites

A FurC position-weight-matrix was generated by MEME (Multiple Em for Motif Elicitation) using the six FurC-DNA binding sequences previously identified in the tested promoter regions of prxA, ftsH, furC, srxA, nifH2 and ahpC genes [7] using the default parameters. This FurC-matrix was used as the input for the software Find Individual Motif Ocurrences (FIMO) (http://meme-suite.org/tools/fimo) [8], to identify the best matches within the Anabaena sp. PCC 7120 genome (Nostoc sp. PCC 7120 = FACHB-418) using a p-value <1x10^-5 as cut-off. Predicted FurC boxes were mapped to annotated CoDing Sequences (CDS) from GenBank assembly (GCA_000009705.1; Sept 2020). Genes with predicted FurC boxes within -1000 bp upstream (intergenic regions) and +50 bp downstream with respect to its start codon were selected as potential FurC targets. In the cases where occurrences were found in the intergenic region of divergently transcribed genes, the gene with closer CDS to the putative FurC-box was chosen as the potential regulated gene. Gene symbol and protein function of the associated genes of the predicted FurC binding sites were annotated according to the databases KEGG (https://www.genome.jp/kegg/) and the information available in literature.

Electrophoretic Mobility Shift Assays (EMSA)

Promoter regions used in the analyses consisted of 250–350 bp DNA fragments containing the predicted FurC binding sites and were obtained by PCR, using the Anabaena sp. PCC 7120 genome as template and the primers included in S1 Table. EMSA analyses were performed with a FurC protein purified as previously described [7]. Reactions for EMSA analyses were performed by mixing increasing concentrations of purified FurC in a final volume of 20 °l with 50 ng of DNA fragments in a binding buffer containing 10 mM Bis Tris-HCl, pH 7.5, 40 mM KCl, 0.1 mg/ml BSA, 1 mM DTT (1,4-dithiothreitol), 100 °M of MnCl₂ and 5% (v/v) glycerol. To assess the specificity of the assay 50 ng of a 150 bp-internal fragment of the gene pkn22 was used as a non-specific competitor The resulting mixture was incubated for 30 min at room temperature. Afterwards DNA fragments were added to the mixture and held for 30 min at room temperature. In all cases the incubated mixtures were loaded into a non-denaturing 6% polyacrylamide gel. Both gel and running buffer included 100 °M MnCl₂. Gels were stained with SYBR ® Safe (Invitrogen) and visualized in a GelDoc 2000 device (Bio-Rad). In all assays, the specific binding to the previously recognized FurC target, hetZ, was used as a positive control.

Cultures and RNA extraction

The RNA samples used for the most of the comparative gene expression experiments performed in this work were extracted and purified as in Sarasa-Buisan et al. 2022 [7]. Briefly, total RNA was prepared from Anabaena sp. PCC 7120 and EB2770FurC strains. The EB2770FurC strain is a furC-overexpressing strain that contains the pAM2770FurC plasmid harboring the furC gene downstream the copper-inducible petE (plastocyanin) promoter [4]. Three independent cultures of each strain were set up by diluting a pre-inoculum from late exponential phase until an OD750 of 0.4 in a final volume of 100 ml in BG11. Cultures were grown in Erlenmeyer flasks on an orbital shaker at 130 rpm and 28°C under a continuous light regime of 30 μmol photons·m^-2·s^-1 and samples were collected after 48 hours (OD₇₅₀ = 0.6–0.7). RNA was extracted and purified from 25 ml of each culture following the method described in Sarasa-Buisan et al. 2022 [7]. In the experiments for measuring hetR expression overtime, the RNA samples used were extracted from cultures set up as stated above and collecting the samples at 1, 6, 11, 24 and 48 hours after the beginning of the experiment. To study differential expression under oxidative stress conditions, RNA samples were obtained from cultures set up as stated above but incubating the cell suspensions at an OD₇₅₀ of 0.4 in the presence and absence of 250 μM H₂O_2. The samples were collected after 1 hour of H₂O₂ treatment. RNA extraction and purification of all experiments were performed from 25 ml of each culture following the method described in Sarasa-Buisan et al. 2022 [7].

Real time RT-PCR assays

The pool of cDNA was synthesized by reverse-transcription of 2 °g of total RNA using SuperScript retrotranscriptase (Invitrogen) following the manufacturer’s conditions. Real time PCR was performed using the ViiA™ 7 Real-Time PCR System (Applied Biosystems). Each reaction was set up by mixing 12.5 °l of SYBR Green PCR Master Mix with 0.4 μl of 25 °M primer mixture and 10 ng of cDNA template in a final volume of 30 °l. Amplification was performed at 60°C. Negative controls with no cDNA were included. The sequences of specific primers of selected genes are defined in S1 Table. Transcript levels of target genes were normalized to those of the housekeeping gene rnpB measured with the same samples [9]. Relative quantification was performed according to the comparative Ct method (ΔΔCt Method) [10]. The minimum fold-change threshold was set up to ± 1.5 fold.

Results

Prediction of novel FurC putative binding sites in the Anabaena sp. PCC 7120 genome

In order to identify potential FurC binding sites, the whole genome of Anabaena sp. PCC 7120 was scanned by a program trained on previously identified sites. The program, FIMO, compares the sequence of a candidate binding site against the nucleotide tendencies at each position of sequences within the training set. Searching of FurC binding sites was performed with a 19-bp FurC-matrix built through MEME software using as input the six sequences closest to a motif found in promoter regions of genes putatively regulated by FurC (prxA, ftsH, furC, srxA, nifH2 and ahpC) [7]. The expression of these genes changed in response to furC overexpression, and their promoter sequences bound FurC in gel shift experiments [3, 4, 7]. The motifs used as a training set as well as the logo built by MEME software are shown in S1 Fig. FIMO analyses found a total of 210 motif occurrences in the genome of Anabaena sp. PCC 7120 (p-value < 1x10^-5) (S2 Table). From the total prediction 55 motif occurrences were found in the putative promoter regions of Anabaena sp. PCC 7120 genome defined as -1000 upstream (intergenic region) and +50 bp downstream the translation start site including two motif occurrences with the closest CDSs corresponding to pseudogenes, whereas interestingly 155 motif occurrences were found outside this region, being 151 located inside the coding regions of genes and 4 located in intergenic convergent regions. The location with respect to the translation start sites of closest CDS is shown in S2 Table. The novel predicted FurC boxes found in the putative promoter regions of genes from Anabaena sp. PCC 7120 with a p-value lower than a 10⁻⁵ threshold are listed in Table 1. In those cases where motif occurrences were found in the intergenic region of divergently transcribed genes, the gene with closer CDS to the putative FurC-box was chosen as the potentially regulated gene for further analyses. All FurC binding sites located in the promoter regions were found in those from chromosomal genes except for two predicted binding sites located in the promoter region of two genes located in the α plasmid of Anabaena sp. PCC 7120, namely asr7140 and asr7090 both encoding both hypothetical proteins. In addition, all the potential FurC targets presented a single predicted FurC binding site in their promoter regions with the exception of all2761 which codes for a hypothetical protein that presents two overlapping predicted FurC boxes. To locate the predicted FurC binding-sites in the promoter regions, the relative position of FurC boxes to the translational start codon (ATG) is shown in Table 1.

Download:

Table 1. Novel predicted FurC-DNA binding sites by FIMO and its potentially regulated gene.

https://doi.org/10.1371/journal.pone.0289761.t001

Determination of FurC binding to predicted sites

The potential binding of FurC to the predicted sites was addressed by performing EMSA assays with all the promoter regions of novel potential FurC targets that presented a predicted FurC-box listed in Table 1 excluding those from genes encoding hypothetical proteins. The EMSA assays showed that among the selected 28 novel potential FurC direct targets, 22 of them were confirmed as actual direct targets of FurC (Fig 1). FurC direct targets were organized in functional categories (Table 2). FurC predicted binding sites were located upstream of genes that take part in a wide range of cellular process such as photosynthesis and photoprotection, oxidative stress, nitrogen metabolism and heterocyst differentiation, central carbon metabolism, zinc homeostasis, amino acid metabolism, as well as regulatory functions, among others. Several EMSA-confirmed FurC predicted binding sites were associated with genes encoding proteins related to photosynthesis and photoprotection. This is consistent with the involvement of FurC in those processes that was described in our previous reported results [4]. Genes as psbZ (asr3992) encoding a PSII structural subunit, fas1 (all3797), an ortholog of fas1 from Anabaena sp. strain L31 that seems to be involved in the response of this cyanobacterium to UV-B radiation stress [11] and all1123, one of the paralogs to the N-terminal domain of the Orange Carotenoid Protein (OCP) present in Anabaena sp. PCC 7120 [12] were direct targets of FurC (Fig 1 and Table 2). OCP proteins interact with the antennas regulating the amount of excitation energy that reaches the reaction centers [13]. However, All1123 seems not to be displaying this particular role in photoprotection, instead it has been suggested that might work as a carotenoid-transport protein [12] or being involved in the response to UV-B stress [14]. Finally, FurC bound to the alr5164 promoter (Fig 1 and Table 2) that correspond to one of the three DegQ protease homologues in Anabaena sp. PCC 7120 [15]. In Synechocystis, DegQ is a protease implicated in the degradation of the PsbO subunit from the PSII [16] and its expression increases under high light conditions [17]. Another EMSA-confirmed FurC predicted binding site corresponded to that located usptream ntrC (alr0737) encoding the NADPH-tiorredoxin reductase C that plays a fundamental role in oxidative stress management and participates in the thermotolerance and regulation of N metabolism [18, 19].

Download:

Fig 1. Validation of the prediction performed by EMSA assays.

The ability of FurC to bind the promoter regions of selected genes was tested. DNA fragments free or mixed with increasing concentrations of recombinant FurC (nM) were separated by 6% PAGE. An internal fragment of the pkn22 gene was used as non-specific competitor DNA.

https://doi.org/10.1371/journal.pone.0289761.g001

Download:

Table 2. Novel putative FurC direct targets found by the genome FurC box search and validated by EMSA.

https://doi.org/10.1371/journal.pone.0289761.t002

Regarding to the “Nitrogen metabolism and heterocyst differentiation” category, FurC was capable of binding to the promoter regions of genes encoding proteins with fundamental roles in the regulation of heterocyst development and nitrogen fixation such as the heterocyst specific ABC transporter, HgdC and, remarkably, the master regulator of the heterocyst differentiation HetR (Fig 1 and Table 2). HgdC takes part of an ABC transporter called Tol-HgdBC located in the cell wall of heterocysts. This transporter participates in the formation of the proper heterocyst glycolipid layer (HGL) in the heterocyst envelope [20]. Interestingly, FurC was able to bind to the hetR promoter. HetR is a key regulator in the heterocyst development and it is required for a suitable pattern formation [21–23]. EMSA assays also indicated that ancrpB (alr2325) is a direct target of FurC. Anabaena sp. PCC 7120 possesses two cAMP receptor proteins called AnCrpA and AnCrpB [24]. AnCrpA regulates the expression of gene clusters relevant in nitrogen fixation processes such as nifB, hglE or coxBII [24]. However, the role of AnCrpB is not that clear because this transcriptional regulator was found to be upregulated in response to rehydration situations [25], presumably controlling the expression of genes involved in the early response to rehydration although it also regulates the expression of some genes related to the response to nitrogen depletion [25].

Moreover, some genes related to carbohydrate metabolism and carbon fixation were verified as FurC direct targets (Fig 1 and Table 2). This is the case of glgP1 (all1272) the glycogen phosphorylase involved in glycogen degradation processes, opcA, the enzyme that activates the glucose 6-phosphate dehydrogenase (G6PDH) in the heterocyst and provides reducing power for the nitrogenase and cmpR (all0862), encoding a LysR type transcriptional regulator that activates the Cmp bicarbonate transport systems in response to low carbon conditions (see below). Another FurC putative binding sites corroborated in vitro were upstream of genes displaying regulatory functions such as the putative transcriptional regulators Asr2041 and Alr3281 and the Alr2137 sensor kinase (Fig 1 and Table 2). Noticeably, another direct target associated with the predicted FurC binding site was the zinc transporter ZupT (all0473) (Fig 1 and Table 2). This gene is the ortholog of zupT from Escherichia coli which encodes a low affinity zinc importer responsible for zinc uptake during zinc-sufficient conditions [26, 27]. Other confirmed FurC targets were genes related to amino acid metabolism, such as alr4124 and pheA and genes encoding proteins displaying other functions such as dnaENI which encodes a DNA polymerase subunit III, alr0252, an Na⁺/H⁺ antiporter, two probable glycosyltransferases all4441 and alr0159, and all4559 encoding a WD-40 repeat protein (Fig 1 and Table 2).

Differential gene expression analysis of newly identified FurC targets in a furC-overexpressing strain versus Anabaena sp. PCC 7120

Transcriptional analyses were conducted to compare the expression levels of the selected novel FurC direct targets in a furC-overexpressing strain (EB2770FurC) versus the wild type strain Anabaena sp. PCC 7120. The furC-overexpressing strain was used instead of a furC knockout mutant because previous attempts to obtain the knockout strain performed by our group and other groups were unsuccessful [3, 4, 7]. Twelve genes were chosen because they were annotated with a specific gene name (glgP1, cmpR, opcA, hetR, ancrpB, hgdC, ntrC, degQ3, psbZ, fas1, pheA, dnaENI and zupT). All the tested genes except for ntrC showed altered expression in the furC-overexpressing strain thus confirming their direct regulation by FurC (Fig 2). Interestingly, most of the selected novel FurC direct targets were overexpressed in the EB2770FurC strain with respect to the wild type Anabaena. Only hetR was slightly downregulated in the furC-overexpressing strain (Fig 2). In the case of glgP1 and fas1 the FurC box is located upstream of the predicted -10 elements as well as in the previously reported FurC direct targets asr1734, ftsH, hepC, rbrA and all3914 [7]. In these cases, FurC could act as a transcriptional activator. On the other hand, transcriptional repressors can act hampering the binding of the RNA polymerase to the -10 element directly or binding downstream the -10 elements [28]. This category would include the previously reported FurC-regulated genes prxA, srxA, CGT3 and psbAIV [7]. Curiously, a large number of sites reported here (opcA, degQ3, pheA, psbZ) sit directly on the start codon of the genes. In these cases, although they are found upregulated in the furC-overexpressing strain, the mechanism exerted by FurC on these genes is still unknown.

Download:

Fig 2. Transcriptional analysis of the novel FurC direct targets in a furC-overexpressing strain and promoter analysis.

(A) Influence of furC overexpression on the mRNA levels of selected genes (see text for choice of genes). Relative Real Time RT-PCR was used. Values are expressed as fold change (EB2770FurC vs. wild-type strain) and correspond to the average of three biological and three technical replicates; the standard deviation is indicated. (B) Graphical representation of the FurC box location in the promoter region of selected genes. The relative positions of FurC boxes related to -10 elements and the tsps reported in Mitschke at al. 2011 [29] are shown. -10 elements are represented as -10 and the distance in bp is indicated. In the case of hetR tsps are retrieved from Buikema and Haselkorn. 2001 [30] and Mitschke et al. 2011 [29].

https://doi.org/10.1371/journal.pone.0289761.g002

FurC could act as transcriptional activator following the canonical MCO mechanism

A big amount of new FurC targets described in the present and our previous work (Sarasa-Buisan et al. 2022) seem to be activated by FurC since their expression increases in the furC- overexpressing strain. In one previous work we demonstrated that when FurC acts as transcriptional repressor, this protein follows the classical MCO mechanism of PerR proteins in vivo [4]. This fact means that when FurC is bound to iron in the presence of hydrogen peroxide, FurC is oxidized and then released from its target promoters allowing the transcription. In the present work we wanted to analyze the mechanism of action in those occasions in which FurC acts as transcriptional activator. The expression of ancrpB, glgP1, cmpR and opcA FurC activated genes and prxA as a control of FurC repressed gene was analyzed after the culture exposure to the presence of 250 μM of hydrogen peroxide for 1 hour. The results are included in Fig 3. As we previously described [4], the expression of prxA is repressed under standard conditions whereas in the presence of hydrogen peroxide the expression is derepressed because FurC was released from the prxA promoter (Fig 3A). In the activated genes ancrpB, cmpR and opcA in which the expression increases in the furC-overexpression strain, the expression decreases in the presence of hydrogen peroxide indicating the releasing of FurC from the promoter and the cease of the activation (Fig 3A and 3B). However, In the case of glgP1 the expression barely change under oxidative stress conditions (Fig 3A). This phenomenon could be explained by the observation made in the well characterized PerR regulon from Bacillus subtilis, in which not all the genes are peroxide regulated, suggesting that PerR protein may exists in distinct Fe or Mn metalated forms that differ in their target selectivity and their sensitivity to oxidation [31].

Download:

Fig 3.

Study of the FurC-mediated activation of gene expression in response to oxidative stress (A) Relative transcription (EB2770FurC vs. Anabaena sp. PCC 7120) of selected genes in presence and absence of the oxidative challenge imposed by H₂O₂ measured by Real Time RT-PCR. Values are expressed as fold change (EB2770FurC vs. wild-type strain) and correspond to the average of three biological and three technical replicates; the standard deviation is indicated. (B) Graphical representation of the FurC-mediated peroxide sensitive mechanism for activation of gene expression. Under oxidative conditions caused by H₂O₂ under iron repletion metal catalyzed oxidation of FurC is produced, ceasing the activation of its targets that occurs in absence of oxidative conditions.

https://doi.org/10.1371/journal.pone.0289761.g003

HetR expression could be controlled by FurC

In the present work we found that FurC was able to bind to the promoter region of hetR suggesting a potential regulation mediated by this transcriptional regulator (Fig 1). Since the FurC box was predicted by FIMO software in the distal region of the hetR upstream region (S1), this was the region firstly analyzed in EMSA assays, yielding a positive result. Afterwards other regions upstream of hetR were also analyzed by EMSA assays with FurC (Fig 4A), indicating binding of FurC to the proximal region as well. The results revealed that FurC was also able to bind the proximal region of the hetR promoter (S3) (Fig 4A). If the hetR promoter region is analyzed in detail, the FurC box is located upstream of -10 element in the distal S1 region, however the existence of a second FurC binding site in the proximal S3 region makes difficult to understand how FurC is regulating the hetR expression (Figs 2B and 4C). Then, the hetR expression was analyzed by Real Time RT-PCR in time-course experiments (1, 6, 11, 24 and 48h) in BG11 medium comparing the EB2770FurC strain with the wild type strain Anabaena sp. PCC 7120 (Fig 4B). We found that the expression of hetR changed along the time in the EB2770FurC strain, being upregulated almost 2-fold after 1h of the beginning of the experiment but downregulated approximately 1.5-fold after 48 h. However, after the first hour, changes in expression are hardly significant. This alteration in the mRNA levels of hetR in the EB2770FurC strain suggests that FurC could be regulating the transcription of hetR in vivo. However, the reason why the hetR expression is first upregulated and subsequently downregulated along the time is unknown. As the genetic background of these strains was identical along the experiment, a possible explanation for this transcriptional change could be that the signals which up to date, have been described to modulate the activity of FurC such as the redox state or metal availability [3, 4, 7] vary across the time in the culture media, leading to changes in the expression profile. Anyway, more experiments must be carried out in the future to clarify this observation.

Download:

Fig 4. Analysis of hetR transcriptional regulation performed by FurC.

(A) Electrophoretic mobility shift assays showing the binding of FurC to the hetR promoter region. The promoter was separated in three DNA regions named S1, S2 and S3. DNA fragments free or mixed with increasing concentrations of recombinant FurC were separated by 6% PAGE. An internal fragment of the pkn22 gene was used as non-specific competitor DNA. (B) Relative transcription of hetR along the time (1, 6, 11, 24 and 48 h) determined by Real-Time RT-PCR in EB2770FurC cells related to Anabaena sp. PCC 7120 ones. Values are expressed as fold change and correspond to the average of three biological and three technical replicates; the SD is indicated. (C) Schematic representation of hetR promoter showing the tsps (+1), -10 elements and FurC box.

https://doi.org/10.1371/journal.pone.0289761.g004

Discussion

An accurate prediction of the FurC/PerR regulon in Anabaena sp. PCC 7120 is essential for our understanding of the scope of the regulation exerted by this protein. Previous work led to the discovery of novel roles of the Peroxide response Regulator beyond the control of oxidative stress management, including the regulation of the photosynthetic metabolism and the heterocyst differentiation [4, 7]. These studies established the selection of new potential targets on the basis of phenotypic observations and transcriptional analysis in a furC overexpressing strain. In the present work an in silico approach has allowed us to obtain a global vision of the extent of the FurC regulon. The bioinformatic search for novel FurC binding sites predicted 210 FurC boxes in the whole genome of Anabaena sp PCC 7120. Interestingly, although it could be considered striking, a considerable number (151) of them were located in internal positions of the CDSs (S2 Table). However, multiple similar cases have been found in literature for other bacterial transcriptional regulators [32–36]. One is the case of the master regulator of nitrogen metabolism NtcA in Anabaena sp. PCC 7120, in which a ChIP-seq analysis identified a high proportion (65%) of NtcA binding regions inside or even downstream of coding regions [36]. More recently, strong pieces of evidence for a meaningful binding of bacterial transcription factors to coding regions have been reported for seven model bacteria, describing three regulatory mechanisms of CDS-bound transcriptional regulators within individual genes, operons, and antisense RNAs [35]. Several possibilities for a specific regulation exerted in these internal sites could be extended for FurC. These possibilities include the potential regulation of internal or antisense transcriptional start sites. This hypothesis would be in agreement with the fact that some of the ORFs that contain the FurC boxes located inside coding regions present either internal or antisense transcriptional start sites [29]. Moreover, a possible role blocking transcription elongation as has been characterized previously for the global transcriptional regulator CodY in B. subtilis [32] could also be possible. Given the complexity for these cases, the present work centered the attention in the FurC boxes found in the putative promoter regions, although we cannot rule out a possible function of FurC outside these regions. If we focus on the predicted FurC binding sites located in putative promoter sequences, among the 28 potential FurC targets selected for EMSA validation, 22 were shown as direct FurC targets. In addition, 13 of them were proved to have their expression altered in a furC-overexpressing strain. Some of these targets displayed key roles in carbon and nitrogen metabolism, photosynthesis and light protection, zinc homeostasis or amino acid metabolism. Carbon and nitrogen metabolisms are tightly coupled in organisms because of a suitable balance of carbon and nitrogen is necessary for optimal growth and proper functioning of the cells [37]. In cyanobacteria, several levels of regulation are superimposed for guarantee the equilibrated uptake and assimilation of nitrogen and carbon sources [37–41]. In Anabaena sp. PCC 7120, many aspects related to the regulation and control of carbon and nitrogen homeostasis are not yet well defined. The engagement of FurC in the regulatory network of heterocyst development and nitrogen fixation was previously suggested. However, in the present work we found that another three genes involved in nitrogen metabolism and heterocyst differentiation (hgdC, ancrpB and hetR) are also direct targets of FurC. Firstly, hgdC encodes a component of Tol-HgdBC transporter which participates in the formation of HGL layer in heterocyst envelopment [20]. Secondly, ancrpB encodes a transcriptional regulator that has been suggested to control the expression of genes related to nitrogen depletion [25]. Surprisingly, FurC was also able to bind to the hetR promoter, hierarchically one of the most important regulators in the process of heterocyst development. Despite of its importance, the transcriptional regulation of HetR is barely understood. Until now, NrrA had been described as a direct positive transcriptional regulator of the HetR expression [42, 43]. Besides, FurA (another member of the FUR family in Anabaena sp. PCC 7120) was also able to bind to the hetR promoter [44]. In addition, some post-transcriptional regulatory mechanisms have been reported for HetR. One of them consists on HetR oligomerization controlled by phosphorylation [45] and the other one defines the interaction with RG(S/T)GR-containing pentapeptides (PatS, PatX and HetN) which act as negative regulators of the HetR activity [46, 47]. In view of this complex panorama, more studies must be carried out to be able to understand the regulation exerted by FurC on the hetR expression.

Regarding to carbon metabolism, our results showed that FurC binds in vitro to the promoters of three important genes comprised in this functional category named glgP1, cmpR and opcA. In addition, the expression of these genes is upregulated in the EB2770FurC strain. These genes display functions that link both carbon and nitrogen metabolisms which are closely intertwined. GlgP1 is a glycogen phosphorylase which catalyzes the generation of glucose-1-phosphate monomers from the glycogen polymer. Anabaena sp. PCC 7120 vegetative cells store carbon belonging to photosynthesis in form of glycogen which is utilized as source of carbon and energy by cyanobacteria during the night. The heterocyst is able to perform photosynthesis (based on PSI) and generate ATP by cyclic photophosphorylation. On the other hand, it has been suggested that vegetative cells could supply carbohydrates (sucrose) to the heterocyst. Some of these carbohydrates derive from glycogen catabolism, indeed NrrA, a nitrogen-regulated response regulator protein, binds to the glgP1 promoter, controlling glycogen metabolism [48, 49]. On the other hand, CmpR is a transcriptional regulator that activates the transcriptional expression of alr2877-alr2880 gene cluster encoding a Cmp bicarbonate transporter under carbon starvation conditions [50]. The Cmp bicarbonate transporter takes part of the carbon concentrating mechanisms developed by cyanobacteria to concentrate carbon in form of CO₂ allowing the efficient actuation of Rubisco in carbon fixation [51]. On the other hand, CmpR has also been proposed as a negative regulator of rbcl operon (encoding Rubisco) [50]. CmpR expression is also regulated by NtcA which again brings into the light the cross-talk between carbon and nitrogen regulatory networks [50]. Finally, in the present work we found that FurC directly controls the expression of OpcA. The redox protein OpcA acts as allosteric activator of glucose-6-phosphate dehydrogenase (G6PDH) [52, 53]. G6PDH is the initial enzyme of the oxidative pentose phosphate pathway in Anabaena sp. PCC 7120 and is essential for nitrogen fixation because this pathway provides NADPH required for the generation of the reducing power for the nitrogenase in heterocysts [54, 55].

Finally, in the present work experiments performed with the furC-overexpressing strain in the presence and absence of hydrogen peroxide revealed a potential activation mechanism that could be performed by FurC (Fig 3B). It can be observed that both the repression and the activation mechanisms are both sensitive to peroxide treatment and may take place depending on the MCO mechanism. This fact is very interesting since the MCO mechanism is a complex mechanism that depends on the oxidative stress found in the cell but also on the iron and manganese availability so that FurC could be regulating its target genes integrating these three different signals. In fact, this mechanism of regulation in response to oxidative stress of FurC might be even more intricated, since a new in vitro regulatory mechanism for FurC activity was recently reported, evidencing that apart from the MCO, FurC undergoes a reversible oxidation through interdimer disulfide bridges formation under mild oxidant conditions and iron deficiency that reversibly prevents the in vitro binding to the DNA [5].

In summary, FurC might be modulating different important processes that are essential for the maintenance of nitrogen and carbon homeostasis in Anabaena sp. PCC 7120 (Fig 5). Previously, we reported that several genes related to heterocyst differentiation and nitrogen fixation were direct targets of FurC (hetZ, asr1734 hepC, nifH, xisHI, rbrA…) [7]. In the present work we found that, FurC controls the heterocyst formation process by regulating the HgdC transporter expression which is involved in the heterocyst envelope formation and FurC could be regulating the expression of hetR, the master regulator of heterocyst development (Fig 5). Furthermore, FurC regulates the expression of CmpR, a transcriptional regulator that controls the carbon fixation process by triggering the expression of bicarbonate transporter Cmp, which is involved in the uptake of bicarbonate into the cell. Additionally, it has been proposed that CmpR could be controlling the expression of Rubisco, enzyme that performs the carbon fixation in the Calvin Cycle (Fig 5). Moreover, FurC controls the expression of GlpP1 glycogen phosphorylase, key enzyme in the catabolism of glycogen and therefore in carbon homeostasis (Fig 5). Finally, FurC seems to upregulate the expression of OpcA, which activates G6PDH in its oxidized form generating reducing power essential for the functioning of nitrogenase in the nitrogen fixation (Fig 5).

Download:

Fig 5. General scheme of the transcriptional regulation performed by FurC of some relevant novel direct target genes involved in the maintenance of nitrogen and carbon homeostasis in Anabaena sp. PCC 7120.

Vegetative cells as well as heterocyst cells are represented to observe processes regulated by FurC in both types of cells. Dashed lines indicate transcriptional regulation, the symbols (+) and (–) show positive and negative transcriptional regulation respectively. CBB cycle: Calvin–Benson–Bassham cycle; TCA cycle: the tricarboxylic acid cycle; 3-PGA: 3-phosphoglycerate; Glc-6P: glucose-6-phosphate; Glc-1P: glucose-1-phosphate; G6PDH: glucose-6-phosphate dehydrogenase.

https://doi.org/10.1371/journal.pone.0289761.g005

Therefore, this work is another piece of evidence that FurC (PerR) is participating in the regulation of the global metabolism of Anabaena sp. PCC 7120, linking the redox state of the cell and the iron and manganese availability with important cellular processes such as photosynthesis, carbon fixation, glycogen catabolism or nitrogen fixation hence confirming the role of FurC (PerR) as a master regulator in Anabaena sp. PCC 7120.

Supporting information

S1 Fig. FurC DNA-binding sequences used as input to build the FurC-matrix by MEME software.

The genes holding these sequences as well as the p-values indicating the degree of similarity with FurC consensus sequence are shown. The logo of the FurC-matrix built by MEME software is included.

https://doi.org/10.1371/journal.pone.0289761.s001

(TIF)

S1 Table. Oligonucleotides used in this study.

https://doi.org/10.1371/journal.pone.0289761.s002

(DOCX)

S2 Table. Predicted FurC-DNA binding sequences found by FIMO in Anabaena sp. PCC 7120 genome.

CoDing Sequence (CDS) annotation according to GenBank assembly (GCA_000009705.1; Sept 2020). Rows with predicted FurC boxes within promoter region defined as -1000 bp upstream (intergenic regions) and +50 bp downstream with respect to the start codon are highlighted in green and the putatively regulated gene is depicted. In case when the FurC box was predicted in the promoter region of two divergently transcribed genes, the two genes are shown. Rows with predicted FurC boxes outside promoter regions are colored in grey and location is indicated. Distant to ATG is defined as number of nucleotides from the translational start site to the closest end position of the predicted FurC box.^a Previously described FurC-DNA binding sites in Sarasa-Buisan et al., 2022 are shown in blue. ^b Motif cooccurrences found downstream coding regions are indicated with (-).

https://doi.org/10.1371/journal.pone.0289761.s003

(XLSX)

S1 Raw images.

https://doi.org/10.1371/journal.pone.0289761.s004

(PDF)

Acknowledgments

Authors thank Dr. Andrés Gonzalez and Dr. Vladimir Espinosa for helpful discussions. We acknowledge the use of Servicio General de Apoyo a la Investigación-SAI, Universidad de Zaragoza.

References

1. Sevilla E, Bes MT, Peleato ML, Fillat MF. Fur-like proteins: Beyond the ferric uptake regulator (Fur) paralog. Arch Biochem Biophys. 2021;701:108770. pmid:33524404
- View Article
- PubMed/NCBI
- Google Scholar
2. Fillat MF. The FUR (ferric uptake regulator) superfamily: diversity and versatility of key transcriptional regulators. Arch Biochem Biophys. 2014;546:41–52. pmid:24513162
- View Article
- PubMed/NCBI
- Google Scholar
3. Yingping F, Lemeille S, Talla E, Janicki A, Denis Y, Zhang CC, et al. Unravelling the cross-talk between iron starvation and oxidative stress responses highlights the key role of PerR (alr0957) in peroxide signalling in the cyanobacterium Nostoc PCC 7120. Environmental microbiology reports. 2014;6(5):468–75.
- View Article
- Google Scholar
4. Sevilla E, Sarasa-Buisan C, Gonzalez A, Cases R, Kufryk G, Peleato ML, et al. Regulation by FurC in Anabaena Links the Oxidative Stress Response to Photosynthetic Metabolism. Plant & cell physiology. 2019;60(8):1778–89.
- View Article
- Google Scholar
5. Sarasa-Buisan C, Emonot E, Martínez-Júlvez M, Sevilla E, Velázquez-Campoy A, Crouzy S, et al. Metal binding and oligomerization properties of FurC (PerR) from Anabaena sp. PCC7120: an additional layer of regulation? Metallomics. 2022;14(10).
- View Article
- Google Scholar
6. Traore DA, El Ghazouani A, Ilango S, Dupuy J, Jacquamet L, Ferrer JL, et al. Crystal structure of the apo-PerR-Zn protein from Bacillus subtilis. Molecular microbiology. 2006;61(5):1211–9.
- View Article
- Google Scholar
7. Sarasa-Buisan C, Guio J, Broset E, Peleato ML, Fillat MF, Sevilla E. FurC (PerR) from Anabaena sp. PCC7120: a versatile transcriptional regulator engaged in the regulatory network of heterocyst development and nitrogen fixation. Environ Microbiol. 2022;24(2):566–82.
- View Article
- Google Scholar
8. Grant CE, Bailey TL, Noble WS. FIMO: scanning for occurrences of a given motif. Bioinformatics. 2011;27(7):1017–8. pmid:21330290
- View Article
- PubMed/NCBI
- Google Scholar
9. Vioque A. Analysis of the gene encoding the RNA subunit of ribonuclease P from cyanobacteria. Nucleic acids research. 1992;20(23):6331–7. pmid:1282240
- View Article
- PubMed/NCBI
- Google Scholar
10. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods. 2001;25(4):402–8. pmid:11846609
- View Article
- PubMed/NCBI
- Google Scholar
11. Babele PK, Singh G, Kumar A, Tyagi MB. Induction and differential expression of certain novel proteins in Anabaena L31 under UV-B radiation stress. Frontiers in microbiology. 2015;6:133.
- View Article
- Google Scholar
12. Lopez-Igual R, Wilson A, Leverenz RL, Melnicki MR, Bourcier de Carbon C, Sutter M, et al. Different Functions of the Paralogs to the N-Terminal Domain of the Orange Carotenoid Protein in the Cyanobacterium Anabaena sp. PCC 7120. Plant Physiol. 2016;171(3):1852–66.
- View Article
- Google Scholar
13. Niyogi KK, Truong TB. Evolution of flexible non-photochemical quenching mechanisms that regulate light harvesting in oxygenic photosynthesis. Current opinion in plant biology. 2013;16(3):307–14. pmid:23583332
- View Article
- PubMed/NCBI
- Google Scholar
14. Shrivastava AK, Chatterjee A, Yadav S, Singh PK, Singh S, Rai LC. UV-B stress induced metabolic rearrangements explored with comparative proteomics in three Anabaena species. Journal of proteomics. 2015;127(Pt A):122–33.
- View Article
- Google Scholar
15. Hahn A, Stevanovic M, Brouwer E, Bublak D, Tripp J, Schorge T, et al. Secretome analysis of Anabaena sp. PCC 7120 and the involvement of the TolC-homologue HgdD in protein secretion. Environ Microbiol. 2015;17(3):767–80.
- View Article
- Google Scholar
16. Roberts IN, Lam XT, Miranda H, Kieselbach T, Funk C. Degradation of PsbO by the Deg protease HhoA Is thioredoxin dependent. PloS one. 2012;7(9):e45713. pmid:23029195
- View Article
- PubMed/NCBI
- Google Scholar
17. Jansen T, Kidron H, Taipaleenmaki H, Salminen T, Maenpaa P. Transcriptional profiles and structural models of the Synechocystis sp. PCC 6803 Deg proteases. Photosynthesis research. 2005;84(1–3):57–63.
- View Article
- Google Scholar
18. Mihara S, Yoshida K, Higo A, Hisabori T. Functional Significance of NADPH-Thioredoxin Reductase C in the Antioxidant Defense System of Cyanobacterium Anabaena sp. PCC 7120. Plant and Cell Physiology. 2016;58(1):86–94.
- View Article
- Google Scholar
19. Deschoenmaeker F, Mihara S, Niwa T, Taguchi H, Wakabayashi KI, Toyoshima M, et al. Thioredoxin pathway in Anabaena sp. PCC 7120: activity of NADPH-thioredoxin reductase C. J Biochem. 2021;169(6):709–19.
- View Article
- Google Scholar
20. Shvarev D, Nishi CN, Wormer L, Maldener I. The ABC Transporter Components HgdB and HgdC are Important for Glycolipid Layer Composition and Function of Heterocysts in Anabaena sp. PCC 7120. Life (Basel). 2018;8(3).
- View Article
- Google Scholar
21. Buikema WJ, Haselkorn R. Characterization of a gene controlling heterocyst differentiation in the cyanobacterium Anabaena 7120. Genes & development. 1991;5(2):321–30.
- View Article
- Google Scholar
22. Buikema WJ, Haselkorn R. Isolation and complementation of nitrogen fixation mutants of the cyanobacterium Anabaena sp. strain PCC 7120. Journal of bacteriology. 1991;173(6):1879–85.
- View Article
- Google Scholar
23. Flores E, Picossi S, Valladares A, Herrero A. Transcriptional regulation of development in heterocyst-forming cyanobacteria. Biochimica et biophysica acta Gene regulatory mechanisms. 2019;1862(7):673–84. pmid:29719238
- View Article
- PubMed/NCBI
- Google Scholar
24. Suzuki T, Yoshimura H, Ehira S, Ikeuchi M, Ohmori M. AnCrpA, a cAMP receptor protein, regulates nif-related gene expression in the cyanobacterium Anabaena sp. strain PCC 7120 grown with nitrate. FEBS letters. 2007;581(1):21–8.
- View Article
- Google Scholar
25. Higo A, Suzuki T, Ikeuchi M, Ohmori M. Dynamic transcriptional changes in response to rehydration in Anabaena sp. PCC 7120. Microbiology (Reading). 2007;153(Pt 11):3685–94.
- View Article
- Google Scholar
26. Nies DH. Zinc starvation response in a cyanobacterium revealed. Journal of bacteriology. 2012;194(10):2407–12. pmid:22389477
- View Article
- PubMed/NCBI
- Google Scholar
27. Grass G, Wong MD, Rosen BP, Smith RL, Rensing C. ZupT is a Zn(II) uptake system in Escherichia coli. Journal of bacteriology. 2002;184(3):864–6.
- View Article
- Google Scholar
28. Payankaulam S, Li LM, Arnosti DN. Transcriptional repression: conserved and evolved features. Current biology : CB. 2010;20(17):R764–71.
- View Article
- Google Scholar
29. Mitschke J, Vioque A, Haas F, Hess WR, Muro-Pastor AM. Dynamics of transcriptional start site selection during nitrogen stress-induced cell differentiation in Anabaena sp. PCC7120. Proceedings of the National Academy of Sciences of the United States of America. 2011;108(50):20130–5.
- View Article
- Google Scholar
30. Buikema WJ, Haselkorn R. Expression of the Anabaena hetR gene from a copper-regulated promoter leads to heterocyst differentiation under repressing conditions. Proceedings of the National Academy of Sciences of the United States of America. 2001;98(5):2729–34.
- View Article
- Google Scholar
31. Fuangthong M, Herbig AF, Bsat N, Helmann JD. Regulation of the Bacillus subtilis fur and perR genes by PerR: not all members of the PerR regulon are peroxide inducible. Journal of bacteriology. 2002;184(12):3276–86.
- View Article
- Google Scholar
32. Belitsky BR, Sonenshein AL. Roadblock repression of transcription by Bacillus subtilis CodY. J Mol Biol. 2011;411(4):729–43.
- View Article
- Google Scholar
33. Jungwirth B, Sala C, Kohl TA, Uplekar S, Baumbach J, Cole ST, et al. High-resolution detection of DNA binding sites of the global transcriptional regulator GlxR in Corynebacterium glutamicum. Microbiology (Reading). 2013;159(Pt 1):12–22.
- View Article
- Google Scholar
34. Perkins TT, Davies MR, Klemm EJ, Rowley G, Wileman T, James K, et al. ChIP-seq and transcriptome analysis of the OmpR regulon of Salmonella enterica serovars Typhi and Typhimurium reveals accessory genes implicated in host colonization. Molecular microbiology. 2013;87(3):526–38.
- View Article
- Google Scholar
35. Hua C, Huang J, Wang T, Sun Y, Liu J, Huang L, et al. Bacterial Transcription Factors Bind to Coding Regions and Regulate Internal Cryptic Promoters. mBio. 2022;13(5):e01643–22. pmid:36200779
- View Article
- PubMed/NCBI
- Google Scholar
36. Picossi S, Flores E, Herrero A. ChIP analysis unravels an exceptionally wide distribution of DNA binding sites for the NtcA transcription factor in a heterocyst-forming cyanobacterium. BMC Genomics. 2014;15(1):22.
- View Article
- Google Scholar
37. Zhang C-C, Zhou C-Z, Burnap RL, Peng L. Carbon/Nitrogen Metabolic Balance: Lessons from Cyanobacteria. Trends in Plant Science. 2018;23(12):1116–30. pmid:30292707
- View Article
- PubMed/NCBI
- Google Scholar
38. Daley SM, Kappell AD, Carrick MJ, Burnap RL. Regulation of the cyanobacterial CO2-concentrating mechanism involves internal sensing of NADP+ and alpha-ketogutarate levels by transcription factor CcmR. PloS one. 2012;7(7):e41286.
- View Article
- Google Scholar
39. Jiang Y-L, Wang X-P, Sun H, Han S-J, Li W-F, Cui N, et al. Coordinating carbon and nitrogen metabolic signaling through the cyanobacterial global repressor NdhR. Proceedings of the National Academy of Sciences. 2018;115(2):403–8. pmid:29279392
- View Article
- PubMed/NCBI
- Google Scholar
40. Nishimura T, Takahashi Y, Yamaguchi O, Suzuki H, Maeda S, Omata T. Mechanism of low CO₂-induced activation of the cmp bicarbonate transporter operon by a LysR family protein in the cyanobacterium Synechococcus elongatus strain PCC 7942. Molecular microbiology. 2008;68(1):98–109.
- View Article
- Google Scholar
41. Herrero A, Muro-Pastor AM, Valladares A, Flores E. Cellular differentiation and the NtcA transcription factor in filamentous cyanobacteria. FEMS microbiology reviews. 2004;28(4):469–87. pmid:15374662
- View Article
- PubMed/NCBI
- Google Scholar
42. Ehira S, Ohmori M. NrrA, a nitrogen-responsive response regulator facilitates heterocyst development in the cyanobacterium Anabaena sp. strain PCC 7120. Molecular microbiology. 2006;59(6):1692–703.
- View Article
- Google Scholar
43. Ehira S, Ohmori M. NrrA directly regulates expression of hetR during heterocyst differentiation in the cyanobacterium Anabaena sp. strain PCC 7120. Journal of bacteriology. 2006;188(24):8520–5.
- View Article
- Google Scholar
44. Gonzalez A, Valladares A, Peleato ML, Fillat MF. FurA influences heterocyst differentiation in Anabaena sp. PCC 7120. FEBS letters. 2013;587(16):2682–90.
- View Article
- Google Scholar
45. Valladares A, Flores E, Herrero A. The heterocyst differentiation transcriptional regulator HetR of the filamentous cyanobacterium Anabaena forms tetramers and can be regulated by phosphorylation. Molecular microbiology. 2016;99(4):808–19.
- View Article
- Google Scholar
46. Khudyakov I, Gladkov G, Elhai J. Inactivation of Three RG(S/T)GR Pentapeptide-Containing Negative Regulators of HetR Results in Lethal Differentiation of Anabaena PCC 7120. Life (Basel). 2020;10(12). pmid:33291589
- View Article
- PubMed/NCBI
- Google Scholar
47. Feldmann EA, Ni S, Sahu ID, Mishler CH, Risser DD, Murakami JL, et al. Evidence for direct binding between HetR from Anabaena sp. PCC 7120 and PatS-5. Biochemistry. 2011;50(43):9212–24.
- View Article
- Google Scholar
48. Ehira S, Ohmori M. NrrA, a nitrogen-regulated response regulator protein, controls glycogen catabolism in the nitrogen-fixing cyanobacterium Anabaena sp. strain PCC 7120. J Biol Chem. 2011;286(44):38109–14.
- View Article
- Google Scholar
49. Ernst A, Reich S, Boger P. Modification of dinitrogenase reductase in the cyanobacterium Anabaena variabilis due to C starvation and ammonia. Journal of bacteriology. 1990;172(2):748–55.
- View Article
- Google Scholar
50. Lopez-Igual R, Picossi S, Lopez-Garrido J, Flores E, Herrero A. N and C control of ABC-type bicarbonate transporter Cmp and its LysR-type transcriptional regulator CmpR in a heterocyst-forming cyanobacterium, Anabaena sp. Environ Microbiol. 2012;14(4):1035–48.
- View Article
- Google Scholar
51. Price GD, Badger MR, Woodger FJ, Long BM. Advances in understanding the cyanobacterial CO2-concentrating-mechanism (CCM): functional components, Ci transporters, diversity, genetic regulation and prospects for engineering into plants. Journal of experimental botany. 2008;59(7):1441–61. pmid:17578868
- View Article
- PubMed/NCBI
- Google Scholar
52. Mihara S, Wakao H, Yoshida K, Higo A, Sugiura K, Tsuchiya A, et al. Thioredoxin regulates G6PDH activity by changing redox states of OpcA in the nitrogen-fixing cyanobacterium Anabaena sp. PCC 7120. The Biochemical journal. 2018;475(6):1091–105.
- View Article
- Google Scholar
53. Mihara S, Sugiura K, Yoshida K, Hisabori T. Thioredoxin targets are regulated in heterocysts of cyanobacterium Anabaena sp. PCC 7120 in a light-independent manner. Journal of experimental botany. 2020;71(6):2018–27.
- View Article
- Google Scholar
54. Winkenbach F, Wolk CP. Activities of enzymes of the oxidative and the reductive pentose phosphate pathways in heterocysts of a blue-green alga. Plant Physiol. 1973;52(5):480–3. pmid:16658588
- View Article
- PubMed/NCBI
- Google Scholar
55. Apte S.K. R, P., and Stewart W.D.P. Electron donation to ferredoxin in heterocysts of the N₂-fixing alga Anabaena cylindrica. Proc R Soc Lond B Biol Sci. 1978;200:1–25.
- View Article
- Google Scholar

[ref1] 1. Sevilla E, Bes MT, Peleato ML, Fillat MF. Fur-like proteins: Beyond the ferric uptake regulator (Fur) paralog. Arch Biochem Biophys. 2021;701:108770. pmid:33524404
View Article
PubMed/NCBI
Google Scholar

[2] View Article

[3] PubMed/NCBI

[4] Google Scholar

[ref2] 2. Fillat MF. The FUR (ferric uptake regulator) superfamily: diversity and versatility of key transcriptional regulators. Arch Biochem Biophys. 2014;546:41–52. pmid:24513162
View Article
PubMed/NCBI
Google Scholar

[6] View Article

[7] PubMed/NCBI

[8] Google Scholar

[ref3] 3. Yingping F, Lemeille S, Talla E, Janicki A, Denis Y, Zhang CC, et al. Unravelling the cross-talk between iron starvation and oxidative stress responses highlights the key role of PerR (alr0957) in peroxide signalling in the cyanobacterium Nostoc PCC 7120. Environmental microbiology reports. 2014;6(5):468–75.
View Article
Google Scholar

[10] View Article

[11] Google Scholar

[ref4] 4. Sevilla E, Sarasa-Buisan C, Gonzalez A, Cases R, Kufryk G, Peleato ML, et al. Regulation by FurC in Anabaena Links the Oxidative Stress Response to Photosynthetic Metabolism. Plant & cell physiology. 2019;60(8):1778–89.
View Article
Google Scholar

[13] View Article

[14] Google Scholar

[ref5] 5. Sarasa-Buisan C, Emonot E, Martínez-Júlvez M, Sevilla E, Velázquez-Campoy A, Crouzy S, et al. Metal binding and oligomerization properties of FurC (PerR) from Anabaena sp. PCC7120: an additional layer of regulation? Metallomics. 2022;14(10).
View Article
Google Scholar

[16] View Article

[17] Google Scholar

[ref6] 6. Traore DA, El Ghazouani A, Ilango S, Dupuy J, Jacquamet L, Ferrer JL, et al. Crystal structure of the apo-PerR-Zn protein from Bacillus subtilis. Molecular microbiology. 2006;61(5):1211–9.
View Article
Google Scholar

[19] View Article

[20] Google Scholar

[ref7] 7. Sarasa-Buisan C, Guio J, Broset E, Peleato ML, Fillat MF, Sevilla E. FurC (PerR) from Anabaena sp. PCC7120: a versatile transcriptional regulator engaged in the regulatory network of heterocyst development and nitrogen fixation. Environ Microbiol. 2022;24(2):566–82.
View Article
Google Scholar

[22] View Article

[23] Google Scholar

[ref8] 8. Grant CE, Bailey TL, Noble WS. FIMO: scanning for occurrences of a given motif. Bioinformatics. 2011;27(7):1017–8. pmid:21330290
View Article
PubMed/NCBI
Google Scholar

[25] View Article

[26] PubMed/NCBI

[27] Google Scholar

[ref9] 9. Vioque A. Analysis of the gene encoding the RNA subunit of ribonuclease P from cyanobacteria. Nucleic acids research. 1992;20(23):6331–7. pmid:1282240
View Article
PubMed/NCBI
Google Scholar

[29] View Article

[30] PubMed/NCBI

[31] Google Scholar

[ref10] 10. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods. 2001;25(4):402–8. pmid:11846609
View Article
PubMed/NCBI
Google Scholar

[33] View Article

[34] PubMed/NCBI

[35] Google Scholar

[ref11] 11. Babele PK, Singh G, Kumar A, Tyagi MB. Induction and differential expression of certain novel proteins in Anabaena L31 under UV-B radiation stress. Frontiers in microbiology. 2015;6:133.
View Article
Google Scholar

[37] View Article

[38] Google Scholar

[ref12] 12. Lopez-Igual R, Wilson A, Leverenz RL, Melnicki MR, Bourcier de Carbon C, Sutter M, et al. Different Functions of the Paralogs to the N-Terminal Domain of the Orange Carotenoid Protein in the Cyanobacterium Anabaena sp. PCC 7120. Plant Physiol. 2016;171(3):1852–66.
View Article
Google Scholar

[40] View Article

[41] Google Scholar

[ref13] 13. Niyogi KK, Truong TB. Evolution of flexible non-photochemical quenching mechanisms that regulate light harvesting in oxygenic photosynthesis. Current opinion in plant biology. 2013;16(3):307–14. pmid:23583332
View Article
PubMed/NCBI
Google Scholar

[43] View Article

[44] PubMed/NCBI

[45] Google Scholar

[ref14] 14. Shrivastava AK, Chatterjee A, Yadav S, Singh PK, Singh S, Rai LC. UV-B stress induced metabolic rearrangements explored with comparative proteomics in three Anabaena species. Journal of proteomics. 2015;127(Pt A):122–33.
View Article
Google Scholar

[47] View Article

[48] Google Scholar

[ref15] 15. Hahn A, Stevanovic M, Brouwer E, Bublak D, Tripp J, Schorge T, et al. Secretome analysis of Anabaena sp. PCC 7120 and the involvement of the TolC-homologue HgdD in protein secretion. Environ Microbiol. 2015;17(3):767–80.
View Article
Google Scholar

[50] View Article

[51] Google Scholar

[ref16] 16. Roberts IN, Lam XT, Miranda H, Kieselbach T, Funk C. Degradation of PsbO by the Deg protease HhoA Is thioredoxin dependent. PloS one. 2012;7(9):e45713. pmid:23029195
View Article
PubMed/NCBI
Google Scholar

[53] View Article

[54] PubMed/NCBI

[55] Google Scholar

[ref17] 17. Jansen T, Kidron H, Taipaleenmaki H, Salminen T, Maenpaa P. Transcriptional profiles and structural models of the Synechocystis sp. PCC 6803 Deg proteases. Photosynthesis research. 2005;84(1–3):57–63.
View Article
Google Scholar

[57] View Article

[58] Google Scholar

[ref18] 18. Mihara S, Yoshida K, Higo A, Hisabori T. Functional Significance of NADPH-Thioredoxin Reductase C in the Antioxidant Defense System of Cyanobacterium Anabaena sp. PCC 7120. Plant and Cell Physiology. 2016;58(1):86–94.
View Article
Google Scholar

[60] View Article

[61] Google Scholar

[ref19] 19. Deschoenmaeker F, Mihara S, Niwa T, Taguchi H, Wakabayashi KI, Toyoshima M, et al. Thioredoxin pathway in Anabaena sp. PCC 7120: activity of NADPH-thioredoxin reductase C. J Biochem. 2021;169(6):709–19.
View Article
Google Scholar

[63] View Article

[64] Google Scholar

[ref20] 20. Shvarev D, Nishi CN, Wormer L, Maldener I. The ABC Transporter Components HgdB and HgdC are Important for Glycolipid Layer Composition and Function of Heterocysts in Anabaena sp. PCC 7120. Life (Basel). 2018;8(3).
View Article
Google Scholar

[66] View Article

[67] Google Scholar

[ref21] 21. Buikema WJ, Haselkorn R. Characterization of a gene controlling heterocyst differentiation in the cyanobacterium Anabaena 7120. Genes & development. 1991;5(2):321–30.
View Article
Google Scholar

[69] View Article

[70] Google Scholar

[ref22] 22. Buikema WJ, Haselkorn R. Isolation and complementation of nitrogen fixation mutants of the cyanobacterium Anabaena sp. strain PCC 7120. Journal of bacteriology. 1991;173(6):1879–85.
View Article
Google Scholar

[72] View Article

[73] Google Scholar

[ref23] 23. Flores E, Picossi S, Valladares A, Herrero A. Transcriptional regulation of development in heterocyst-forming cyanobacteria. Biochimica et biophysica acta Gene regulatory mechanisms. 2019;1862(7):673–84. pmid:29719238
View Article
PubMed/NCBI
Google Scholar

[75] View Article

[76] PubMed/NCBI

[77] Google Scholar

[ref24] 24. Suzuki T, Yoshimura H, Ehira S, Ikeuchi M, Ohmori M. AnCrpA, a cAMP receptor protein, regulates nif-related gene expression in the cyanobacterium Anabaena sp. strain PCC 7120 grown with nitrate. FEBS letters. 2007;581(1):21–8.
View Article
Google Scholar

[79] View Article

[80] Google Scholar

[ref25] 25. Higo A, Suzuki T, Ikeuchi M, Ohmori M. Dynamic transcriptional changes in response to rehydration in Anabaena sp. PCC 7120. Microbiology (Reading). 2007;153(Pt 11):3685–94.
View Article
Google Scholar

[82] View Article

[83] Google Scholar

[ref26] 26. Nies DH. Zinc starvation response in a cyanobacterium revealed. Journal of bacteriology. 2012;194(10):2407–12. pmid:22389477
View Article
PubMed/NCBI
Google Scholar

[85] View Article

[86] PubMed/NCBI

[87] Google Scholar

[ref27] 27. Grass G, Wong MD, Rosen BP, Smith RL, Rensing C. ZupT is a Zn(II) uptake system in Escherichia coli. Journal of bacteriology. 2002;184(3):864–6.
View Article
Google Scholar

[89] View Article

[90] Google Scholar

[ref28] 28. Payankaulam S, Li LM, Arnosti DN. Transcriptional repression: conserved and evolved features. Current biology : CB. 2010;20(17):R764–71.
View Article
Google Scholar

[92] View Article

[93] Google Scholar

[ref29] 29. Mitschke J, Vioque A, Haas F, Hess WR, Muro-Pastor AM. Dynamics of transcriptional start site selection during nitrogen stress-induced cell differentiation in Anabaena sp. PCC7120. Proceedings of the National Academy of Sciences of the United States of America. 2011;108(50):20130–5.
View Article
Google Scholar

[95] View Article

[96] Google Scholar

[ref30] 30. Buikema WJ, Haselkorn R. Expression of the Anabaena hetR gene from a copper-regulated promoter leads to heterocyst differentiation under repressing conditions. Proceedings of the National Academy of Sciences of the United States of America. 2001;98(5):2729–34.
View Article
Google Scholar

[98] View Article

[99] Google Scholar

[ref31] 31. Fuangthong M, Herbig AF, Bsat N, Helmann JD. Regulation of the Bacillus subtilis fur and perR genes by PerR: not all members of the PerR regulon are peroxide inducible. Journal of bacteriology. 2002;184(12):3276–86.
View Article
Google Scholar

[101] View Article

[102] Google Scholar

[ref32] 32. Belitsky BR, Sonenshein AL. Roadblock repression of transcription by Bacillus subtilis CodY. J Mol Biol. 2011;411(4):729–43.
View Article
Google Scholar

[104] View Article

[105] Google Scholar

[ref33] 33. Jungwirth B, Sala C, Kohl TA, Uplekar S, Baumbach J, Cole ST, et al. High-resolution detection of DNA binding sites of the global transcriptional regulator GlxR in Corynebacterium glutamicum. Microbiology (Reading). 2013;159(Pt 1):12–22.
View Article
Google Scholar

[107] View Article

[108] Google Scholar

[ref34] 34. Perkins TT, Davies MR, Klemm EJ, Rowley G, Wileman T, James K, et al. ChIP-seq and transcriptome analysis of the OmpR regulon of Salmonella enterica serovars Typhi and Typhimurium reveals accessory genes implicated in host colonization. Molecular microbiology. 2013;87(3):526–38.
View Article
Google Scholar

[110] View Article

[111] Google Scholar

[ref35] 35. Hua C, Huang J, Wang T, Sun Y, Liu J, Huang L, et al. Bacterial Transcription Factors Bind to Coding Regions and Regulate Internal Cryptic Promoters. mBio. 2022;13(5):e01643–22. pmid:36200779
View Article
PubMed/NCBI
Google Scholar

[113] View Article

[114] PubMed/NCBI

[115] Google Scholar

[ref36] 36. Picossi S, Flores E, Herrero A. ChIP analysis unravels an exceptionally wide distribution of DNA binding sites for the NtcA transcription factor in a heterocyst-forming cyanobacterium. BMC Genomics. 2014;15(1):22.
View Article
Google Scholar

[117] View Article

[118] Google Scholar

[ref37] 37. Zhang C-C, Zhou C-Z, Burnap RL, Peng L. Carbon/Nitrogen Metabolic Balance: Lessons from Cyanobacteria. Trends in Plant Science. 2018;23(12):1116–30. pmid:30292707
View Article
PubMed/NCBI
Google Scholar

[120] View Article

[121] PubMed/NCBI

[122] Google Scholar

[ref38] 38. Daley SM, Kappell AD, Carrick MJ, Burnap RL. Regulation of the cyanobacterial CO2-concentrating mechanism involves internal sensing of NADP+ and alpha-ketogutarate levels by transcription factor CcmR. PloS one. 2012;7(7):e41286.
View Article
Google Scholar

[124] View Article

[125] Google Scholar

[ref39] 39. Jiang Y-L, Wang X-P, Sun H, Han S-J, Li W-F, Cui N, et al. Coordinating carbon and nitrogen metabolic signaling through the cyanobacterial global repressor NdhR. Proceedings of the National Academy of Sciences. 2018;115(2):403–8. pmid:29279392
View Article
PubMed/NCBI
Google Scholar

[127] View Article

[128] PubMed/NCBI

[129] Google Scholar

[ref40] 40. Nishimura T, Takahashi Y, Yamaguchi O, Suzuki H, Maeda S, Omata T. Mechanism of low CO₂-induced activation of the cmp bicarbonate transporter operon by a LysR family protein in the cyanobacterium Synechococcus elongatus strain PCC 7942. Molecular microbiology. 2008;68(1):98–109.
View Article
Google Scholar

[131] View Article

[132] Google Scholar

[ref41] 41. Herrero A, Muro-Pastor AM, Valladares A, Flores E. Cellular differentiation and the NtcA transcription factor in filamentous cyanobacteria. FEMS microbiology reviews. 2004;28(4):469–87. pmid:15374662
View Article
PubMed/NCBI
Google Scholar

[134] View Article

[135] PubMed/NCBI

[136] Google Scholar

[ref42] 42. Ehira S, Ohmori M. NrrA, a nitrogen-responsive response regulator facilitates heterocyst development in the cyanobacterium Anabaena sp. strain PCC 7120. Molecular microbiology. 2006;59(6):1692–703.
View Article
Google Scholar

[138] View Article

[139] Google Scholar

[ref43] 43. Ehira S, Ohmori M. NrrA directly regulates expression of hetR during heterocyst differentiation in the cyanobacterium Anabaena sp. strain PCC 7120. Journal of bacteriology. 2006;188(24):8520–5.
View Article
Google Scholar

[141] View Article

[142] Google Scholar

[ref44] 44. Gonzalez A, Valladares A, Peleato ML, Fillat MF. FurA influences heterocyst differentiation in Anabaena sp. PCC 7120. FEBS letters. 2013;587(16):2682–90.
View Article
Google Scholar

[144] View Article

[145] Google Scholar

[ref45] 45. Valladares A, Flores E, Herrero A. The heterocyst differentiation transcriptional regulator HetR of the filamentous cyanobacterium Anabaena forms tetramers and can be regulated by phosphorylation. Molecular microbiology. 2016;99(4):808–19.
View Article
Google Scholar

[147] View Article

[148] Google Scholar

[ref46] 46. Khudyakov I, Gladkov G, Elhai J. Inactivation of Three RG(S/T)GR Pentapeptide-Containing Negative Regulators of HetR Results in Lethal Differentiation of Anabaena PCC 7120. Life (Basel). 2020;10(12). pmid:33291589
View Article
PubMed/NCBI
Google Scholar

[150] View Article

[151] PubMed/NCBI

[152] Google Scholar

[ref47] 47. Feldmann EA, Ni S, Sahu ID, Mishler CH, Risser DD, Murakami JL, et al. Evidence for direct binding between HetR from Anabaena sp. PCC 7120 and PatS-5. Biochemistry. 2011;50(43):9212–24.
View Article
Google Scholar

[154] View Article

[155] Google Scholar

[ref48] 48. Ehira S, Ohmori M. NrrA, a nitrogen-regulated response regulator protein, controls glycogen catabolism in the nitrogen-fixing cyanobacterium Anabaena sp. strain PCC 7120. J Biol Chem. 2011;286(44):38109–14.
View Article
Google Scholar

[157] View Article

[158] Google Scholar

[ref49] 49. Ernst A, Reich S, Boger P. Modification of dinitrogenase reductase in the cyanobacterium Anabaena variabilis due to C starvation and ammonia. Journal of bacteriology. 1990;172(2):748–55.
View Article
Google Scholar

[160] View Article

[161] Google Scholar

[ref50] 50. Lopez-Igual R, Picossi S, Lopez-Garrido J, Flores E, Herrero A. N and C control of ABC-type bicarbonate transporter Cmp and its LysR-type transcriptional regulator CmpR in a heterocyst-forming cyanobacterium, Anabaena sp. Environ Microbiol. 2012;14(4):1035–48.
View Article
Google Scholar

[163] View Article

[164] Google Scholar

[ref51] 51. Price GD, Badger MR, Woodger FJ, Long BM. Advances in understanding the cyanobacterial CO2-concentrating-mechanism (CCM): functional components, Ci transporters, diversity, genetic regulation and prospects for engineering into plants. Journal of experimental botany. 2008;59(7):1441–61. pmid:17578868
View Article
PubMed/NCBI
Google Scholar

[166] View Article

[167] PubMed/NCBI

[168] Google Scholar

[ref52] 52. Mihara S, Wakao H, Yoshida K, Higo A, Sugiura K, Tsuchiya A, et al. Thioredoxin regulates G6PDH activity by changing redox states of OpcA in the nitrogen-fixing cyanobacterium Anabaena sp. PCC 7120. The Biochemical journal. 2018;475(6):1091–105.
View Article
Google Scholar

[170] View Article

[171] Google Scholar

[ref53] 53. Mihara S, Sugiura K, Yoshida K, Hisabori T. Thioredoxin targets are regulated in heterocysts of cyanobacterium Anabaena sp. PCC 7120 in a light-independent manner. Journal of experimental botany. 2020;71(6):2018–27.
View Article
Google Scholar

[173] View Article

[174] Google Scholar

[ref54] 54. Winkenbach F, Wolk CP. Activities of enzymes of the oxidative and the reductive pentose phosphate pathways in heterocysts of a blue-green alga. Plant Physiol. 1973;52(5):480–3. pmid:16658588
View Article
PubMed/NCBI
Google Scholar

[176] View Article

[177] PubMed/NCBI

[178] Google Scholar

[ref55] 55. Apte S.K. R, P., and Stewart W.D.P. Electron donation to ferredoxin in heterocysts of the N₂-fixing alga Anabaena cylindrica. Proc R Soc Lond B Biol Sci. 1978;200:1–25.
View Article
Google Scholar

[180] View Article

[181] Google Scholar

Figures

Abstract

Introduction

Materials and methods

Bioinformatic prediction of FurC putative binding sites

Electrophoretic Mobility Shift Assays (EMSA)

Cultures and RNA extraction

Real time RT-PCR assays

Results

Prediction of novel FurC putative binding sites in the Anabaena sp. PCC 7120 genome

Determination of FurC binding to predicted sites

Differential gene expression analysis of newly identified FurC targets in a furC-overexpressing strain versus Anabaena sp. PCC 7120

FurC could act as transcriptional activator following the canonical MCO mechanism

HetR expression could be controlled by FurC

Discussion

Supporting information

S1 Fig. FurC DNA-binding sequences used as input to build the FurC-matrix by MEME software.

S1 Table. Oligonucleotides used in this study.

S2 Table. Predicted FurC-DNA binding sequences found by FIMO in Anabaena sp. PCC 7120 genome.

S1 Raw images.

Acknowledgments

References