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1 Department of Biochemistry and Molecular and Cell Biology, University of Zaragoza, Zaragoza, Spain
2 Biocomputation and Complex Systems Physics Institute (BiFi), University of Zaragoza, Zaragoza, Spain
3 MSU-DOE Plant Research Laboratory and Department of Plant Biology, Michigan State University, East Lansing, MI, USA
Correspondence
M. F. Fillat
fillat{at}unizar.es
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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-furA.
A supplementary figure showing the construction of plasmids is available with the online version of this paper.
These authors contributed equally to this work.
Present address: Plant and Microbial Biology Department, 211 Koshland Hall, University of California at Berkeley, 94720 Berkeley, CA, USA.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). In cyanobacteria, iron availability critically influences rates of photosynthesis and nitrogen fixation (Straus, 1994; Kustka et al., 2002). To overcome iron stress, prokaryotes have evolved a number of responses, most of them under genetic control by the Fur (ferric uptake regulator) proteins. These global regulatory proteins are involved in the maintenance of iron homeostasis. They recognize specific DNA sequences denoted iron boxes, and coordinate iron metabolism with cellular defences against several stresses (Andrews et al., 2003). It is thought that Fur proteins act as classical repressors. Under iron-rich conditions, Fur dimers complexed with iron(II) bind to iron boxes, preventing transcription (Bagg & Neilands, 1987). Fur proteins are constitutive, and are highly abundant compared to other regulators (Zheng et al., 1999). The genome of Anabaena sp. strain PCC 7120 (hereinafter, Anabaena sp.) encodes three Fur homologues. FurA (the product of ORF all1691) shows the highest homology with Fur from heterotrophic bacteria (Hernández et al., 2004a) and is an essential protein in cyanobacteria (Hernández et al., 2006a). The amount of active FurA in Anabaena sp. responds to a complex balance between iron availability, redox status of the cell and expression of the antisense RNA -furA, among other effectors (Hernández et al., 2004a, 2006a).
In the absence of combined nitrogen, Anabaena sp. is able to reduce atmospheric dinitrogen to ammonium in a process that involves activation of numerous genes. Since nitrogenase is very sensitive to oxygen, nitrogen-fixing cyanobacteria have developed a variety of strategies to allow oxygenic photosynthesis and nitrogen fixation to take place simultaneously. In Anabaena sp., nitrogen fixation is confined to specialized cells called heterocysts. To protect nitrogenase from inactivation by oxygen, the heterocyst lacks photosystem II activity, has an elevated rate of respiration and has a thick envelope that minimizes the influx of O2, thus creating a micro-oxic intracellular environment (Wolk et al., 1994). Some proteins involved in heterocyst development and metabolism, including PatB (Liang et al., 1993), FdxH (Böhme & Haselkorn, 1988) and the constituent proteins of nitrogenase and of the up-regulated respiratory apparatus, are rich in iron. Therefore, the differentiating cells must use much of their iron reserves to construct the machinery required for nitrogen fixation and maintenance of micro-oxic internal conditions.
Surprisingly little is known about the regulation of nitrogen fixation genes. NtcA, the principal transcription factor involved in the regulatory network of nitrogen metabolism, belongs to the CAP family (Herrero et al., 2001). NtcA helps to regulate the initiation and progression of heterocyst differentiation, and is also involved in the activation of numerous genes involved in nitrogen assimilation. NtcA senses the nitrogen status in cyanobacteria via 2-oxoglutarate (Laurent et al., 2005) and positively regulates genes by binding to their promoter regions at sites that exhibit the consensus sequence GTAN8TAC. By binding to target sites that are located downstream of the transcription start point (tsp), NtcA can also play a role as repressor (Herrero et al., 2001; Muro-Pastor et al., 2001). It has been proposed that NtcA responds to redox changes (Jiang et al., 1997; Alfonso et al., 2001). Moreover, NtcA may coordinate iron acquisition and nitrogen metabolism by activating the expression of pkn41 and pkn42 (Cheng et al., 2006).
We show here that the nitrogen status of a culture contributes to the regulation of the expression of FurA. Nitrogen deprivation strongly activated PfurA in proheterocysts and heterocysts. In contrast, fusions of reporter genes to PfurB and PfurC showed no changes in expression in response to nitrogen deprivation and heterocyst differentiation. NtcA, in addition to other factors previously reported (Bes et al., 2001; Hernández et al., 2004a, 2006a), is involved in the regulation of expression of furA, as illustrated by great differences in the transcriptional pattern of the furA locus in an ntcA mutant. These results suggest that FurA plays a significant role in the metabolism of heterocysts.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). Cells were grown at 30 °C in nitrogen-free medium BG-110 or in BG-11 medium supplemented with 6 mM NH4Cl plus 12 mM TES/NaOH buffer (pH 7.5) (Rippka, 1988). Cultures of CSE2 were supplemented with 2 µg ml–1 each of streptomycin and spectinomycin. Escherichia coli strains DH5 and BL21-Gold DE3 grown in Luria–Bertani liquid or agar medium at 37 °C were used for DNA manipulations. Media were supplemented with appropriate antibiotics according to standard protocols (Sambrook et al., 1989). To place cultures under nitrogen-stress conditions, cells from a nitrogen-replete medium were washed three times at room temperature with, and resuspended in, BG110C medium (i.e., BG110 supplemented with 10 mM NaHCO3), and incubated under growth conditions for the times indicated.
RNA isolation and Northern analysis.
Anabaena sp. RNA was prepared as described previously (Hernández et al., 2006a), using 40 ml cells containing 
5 µg chlorophyll a ml–1. Northern blot analysis was performed using 40 µg RNA, as described by Hernández et al. (2006a). DNA probes for Northern blotting were obtained by PCR amplification of the furA sequence using the primers listed in Table 1. Transcripts were quantified using the Cyclone Storage Phosphor System (Packard).
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, were cloned as 338, 457 and 413 bp fragments into pGEM-T (Promega), producing pCZS3, pCZS4 and pCZS5, respectively. Promoterless gfpmut2 was cloned as a 911 bp XbaI–PstI fragment from pGFPM2 (Cormack et al., 1996) between the SpeI and PstI sites of pCZS3, pCZS4 or pCZS5, producing pCZS9, pCZS10 and pCZS11, respectively (see supplementary Fig. S1, available with the online version of this paper). Digestion of the resulting constructs with SacII and PstI released Pfur-gfp on fragments that were ligated into SacII- and PstI-digested pRL2696, producing pCZS25, pCZS26 and pCZS27, respectively. pRL2696 consists of the RSF1010-based vector pRL1383a (Fan et al., 2005) into whose unique SalI site is inserted cassette C.C1 from pRL178 (Elhai & Wolk, 1988a). Control plasmid pCZS20 (Fig. S1) was constructed by cloning promoterless gfp as an XbaI–PstI fragment from pGFPM2 into pRL2696 digested with XbaI and PstI. pCZS20, pCZS25, pCZS26 and pCZS27 were introduced into Anabaena sp. by conjugation (Elhai & Wolk, 1988b). Photographs were taken with a Nikon DXM1200F digital camera mounted on a Nikon Eclipse50i microscope. GFP expression was visualized by fluorescence microscopy, with a Nikon Epi-fluorescence filter B-2A (EX 450-490, DM 505, BA520). Images were captured with ACT-1 and edited with Adobe Photoshop 7.0.
Electrophoretic mobility shift assays (EMSAs).
DNA fragments of 389 and 307 bp that contain the promoter regions of furA, and a 420 bp fragment with the promoter of the alr1690--furA RNA transcript (Table 1
), were amplified by PCR and used in non-radioactive band-shift assays, as described previously (Bes et al., 2001). Binding assays were carried out in a final volume of 20 µl containing 10 mM Bistris/HCl (pH 7.5), 40 mM KCl, 1 mM dithiothreitol, 0.1 mM MgCl2, 0.05 µg bovine serum albumin µl–1, 75–100 ng of the DNA fragment to be tested, 75 ng of a control DNA fragment and 5 % (v/v) glycerol. The DNA fragment used as unrelated, control DNA was a 228 bp fragment from human apoE (Bes et al., 2001). Assays were carried out with 5 pmol histidine-tagged NtcA purified from E. coli cells bearing pCSAM70 (Muro-Pastor et al., 1999). Results were processed with an Imagestore 5000 image analyser (UVP Inc.).
DNase I footprinting.
The DNase I protection assay used was modified from that described previously (Frías et al., 2000). The PCR product containing the promoter region of furA used in the EMSAs was cloned into pGEM-T and sequenced to verify the fidelity of the amplification. The fragment excised using enzymes NcoI and PstI was 3'-end-labelled with the Klenow fragment of DNA polymerase and [-32P]dCTP (3000 Ci mmol–1; 111 TBq mmol–1). Binding reactions were performed in a volume of 20 µl, with incubation of 0.05 pmol labelled promoter with 1 µM purified NtcA in the presence of 1.5 µg salmon sperm DNA as non-specific competitor. Radioactive bands were visualized with a Cyclone storage phosphor system and OptiQuant image analysis software (Packard).
Electrophoresis and immunoblotting.
Crude extracts were prepared by ultrasonic treatment, followed by centrifugation to remove cell debris. Protein was quantified using a bicinchoninic acid protein assay reagent (Pierce). Samples (25 µg) of total protein were separated by SDS-PAGE (17 % polyacrylamide gel). For immunoblotting, proteins were transferred by electrophoresis to PVDF (0.45 mm pore size transfer membrane; Waters), as described previously (Hernández et al., 2006a). Rabbit polyclonal antibodies raised against Anabaena sp. FurA protein were used, and the blot was visualized and quantified with a Gel Doc 2000 image analyser (Bio-Rad).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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-furA transcripts was performed using RNA from cultures grown with ammonium and then deprived of nitrogen. The results shown in Fig. 1(a) indicate that under nitrogen-fixing conditions, the abundance of the 0.6 kb furA transcript increased 2.3-fold with respect to the level of message detected in the presence of ammonium, whereas the amount of the 2.2 kb alr1690--furA RNA decreased by 30 %. As a result, FurA expression increased, as detected by Western blot analysis (Fig. 1c).
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-furA. The signature GTAN8TAC, usually flanked by A/T nucleotides, is strongly conserved in the promoters of NtcA-activated genes. However, the promoters of a number of nitrogen-assimilation genes have been reported that resemble, but do not match, the canonical consensus (Herrero et al., 2001) shown in Fig. 2. Promoters of furA and of alr1690--furA have been defined previously by determination of their transcription start points (Hernández et al., 2006a, b). Sequences present in the promoter regions of furA and alr1690--furA that are similar to the canonical targets of NtcA are shown in Fig. 2. PfurA exhibits three imperfect NtcA sites centred at bp –115.5, –92.5 and –40.5 with respect to its tsp. A fourth sequence that matches the NtcA-binding consensus includes the translation start codon and part of the coding sequence. Similarly, the alr1690--furA promoter contains two sequences, at –146.5 and +0.5, that resemble NtcA-binding sites. The upstream sequences are similar to NtcA-activated NtcA-boxes, whereas the downstream sequence, which includes the tsp, could, depending on the nitrogen status of the cell, bind NtcA and act as a repressor. To verify whether NtcA is responsible for the increase in the expression of furA under conditions of nitrogen deprivation, we carried out Northern and Western analyses of cultures of the CSE2 strain subjected to nitrogen step-down. In all cases tested, Northern analysis of CSE2 cells grown in the presence of ammonium showed higher transcription of the furA message than that in the wild-type strain, in agreement with the amounts of FurA detected by Western analysis, which were around 1.3- to 1.5-fold higher in the CSE2 strain (lanes 
from Fig. 1c). However, changes in the expression of FurA in CSE2 cultures in response to nitrogen step-down were much less than those in the wild-type strain. In the ntcA mutant, the 2.2 kb transcript dropped dramatically, while a 0.35 kb message appeared. Because non-specific hybridization of the furA probe with furB and furC homologues has been ruled out (Hernández et al., 2006a), the 0.35 kb message may result from processing of the alr1690--furA transcript in CSE2 cells. Alternatively, transcription of alr1690--furA may be repressed in the ntcA mutant, while a new 0.35 kb message is transcribed.
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-furA-furA. Since the region of PfurA that showed the highest homology to the canonical NtcA box contains the translation start point, binding assays were performed with two DNA fragments, PfurA1, which spans positions –331 to +58 with respect to the tsp, and a shorter fragment, PfurA2, which spans positions –331 to –25 and lacks the ATG. When NtcA was incubated with either PfurA1 or PfurA2, at least two NtcA–DNA complexes were detected (Fig. 3, lanes 3 and 6). Mobility-shift assays of Palr1690--furA showed that both FurA and NtcA bind to this DNA. FurA shifted only a small amount of the promoter fragment, whereas two NtcA–Palr1690--furA complexes were clearly observable.
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(a) shows several NtcA-protected regions of nucleotides located in two main regions of PfurA, approximately from nt –122 to –100 and from nt –47 to nt +57. The protected nucleotides are identified in Fig. 4(b). All are located in the putative NtcA-binding sites depicted in Fig. 2. The protected G and C located at –47 and –34 bp, respectively, relative to the tsp are located in the imperfect NtcA binding site centred at –40.5. This site forms a perfect palindrome and is accompanied by the motif TAN3T 20 nucleotides downstream of the NtcA signature, in good accord with the location of such a motif relative to canonical NtcA-activated promoters. This site is part of one of the two regions protected by FurA in DNase I footprinting experiments described by Hernández et al. (2006b). Perhaps this part of the promoter is occupied either by FurA or by NtcA depending on the relative abundance of the pools of iron and combined nitrogen. Binding of NtcA to nucleotides that span the translation start site would allow down-regulation of expression of furA under certain conditions. Such binding may account for the observation that in ammonium-containing media, where only vegetative cells are present, strain CSE2 produces more FurA than do cells of wild-type Anabaena sp. Our in vitro results show that NtcA can recognize and bind to NtcA-like signatures present in PfurA.
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), primer extension studies were carried out using the RNAs isolated from cultures of both Anabaena sp. and the CSE2 strain grown both in the presence of ammonium and submitted to nitrogen step-down. In all cases tested, only the tsp previously reported was detected (not shown).
furA is strongly induced in proheterocysts and heterocysts
To find out whether the increase in the expression of furA in the absence of fixed nitrogen was a general response produced throughout filaments of Anabaena sp. or was restricted to the heterocysts, expression from PfurA was investigated using Gfp as reporter (Fig. 5). Because nitrogen status does not affect transcription levels of furB and furC (as determined by Northern analysis; data not shown), we used, as controls, not only the plasmid pCZ20 which bears a ‘non-promoted gfp’, but also pCZ20-based vectors in which PfurB and PfurC were added to drive gfp. Recombinant Anabaena sp. bearing pCZS20, pCZS25, pCZS26 or pCZS27 was examined by fluorescence microscopy. The experiments were performed three times with independently grown cultures. Mature heterocysts were observed by light microscopy in recombinant Anabaena sp. strains 24 h after nitrogen step-down. We first examined the expression of PfurA. When cells were grown in the presence of ammonium, a faint fluorescence due to the expression of furA in vegetative cells was detected (Fig. 5a). A slight induction of Gfp from PfurA at regular intervals along the filament was observed after 15 h of nitrogen deprivation. Fluorescence reached its maximum by 24 h and remained approximately constant in mature heterocysts for at least 6 more days of nitrogen deprivation. Thus, furA is induced in proheterocysts and remains stably expressed in mature heterocysts. Fig. 5(c) shows that expression of Gfp from PfurC was nearly the same in heterocysts and vegetative cells. We were unable, at the same level of sensitivity, to detect expression of PfurB by fluorescence microscopy (Fig. 5b), but by increasing the sensitivity, we could detect faint fluorescence that was also of nearly equal intensity in the two types of cells. Because no gfp-based fluorescence was observed with pCZS20 (which contains promoterless gfp; Fig. 5d), the observed fluorescence resulted from the activity of the fur promoters.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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-furA antisense RNA interferes with the furA transcript. In vitro analysis shows that binding of FurA to its DNA targets is modulated by several parameters such as the redox potential, the availability of metal, and the presence of haem (Hernández et al., 2006b, 2004b).
In this work, we show that nitrogen status also modulates the expression of furA. The use of a Gfp reporter fused to the promoters of the three fur homologues showed that PfurA is strongly activated in proheterocysts and remains overexpressed in the mature heterocyst. However, no changes were observed in the levels of expression of furB and furC. NtcA binds to PfurA and to the promoter of the dicistron alr1690--furA, whose transcription is severely affected in an ntcA mutant. A search for NtcA boxes in the promoter regions of both messages showed the presence of several putative NtcA-binding sites. Binding and footprinting assays showed that, in vitro, NtcA can bind to sites in both promoters. Similar observations have been made with the NtcA-dependent promoters of nir and xisA (Chastain et al., 1990; Frías et al., 2000). Like other regulators of the CAP family, NtcA appears to act either as an activator or as a repressor depending on the metabolic status of the cells (Kolb et al., 1993; Herrero et al., 2001). In the case of alr1690--furA, NtcA would act as a repressor of the alr1690--furA message if it binds to the box that covers the tsp of this dicistron (Fig. 2). However, binding of NtcA to the site centred at –146.5 would activate transcription of that message. The structure of PfurA, which shows three imperfect NtcA-binding sites centred at –115.5, –92.5 and –40.5, is more complex. The last of these three NtcA signatures is located at the canonical distance observed in class II CAP-activated promoters (Busby & Ebright, 1997) and overlaps with the site II of FurA-protected regions (Hernández et al., 2006b). The convergence of several transcriptional regulators in the promoter region of fur has been demonstrated in several micro-organisms (Zheng et al., 1999; Fuangthong et al., 2002). In E. coli, in addition to autoregulation by its product, transcription of fur is modulated by OxyR and SoxRS, the primary regulators of responses to oxidative stress (Zheng et al., 1999). Moreover, transcription of fur is stimulated by the cAMP–CAP system (De Lorenzo et al., 1988), and tight functional interactions between Fur and CRP have been confirmed using whole-transcriptome approaches (Zhang et al., 2005).
In cyanobacteria, many iron-responsive genes, such as nifJ, nblA, petH, and the putatively kinase-encoding genes pkn41 and pkn42, are also modulated by NtcA (Valladares et al., 1999; Luque et al., 2001; Cheng et al., 2006), suggesting that NtcA plays a key role in the coordination of iron acquisition and nitrogen metabolism. Interaction of FurA and NtcA at their overlapping binding sites in PfurA would result in integration of metabolic signals that involve C/N ratio, redox status, iron availability and/or ATP levels. We hypothesize that the enhanced expression of furA in the heterocysts of wild-type Anabaena sp., compared with the negligible increase in expression of the regulatory gene in the ntcA strain CSE2, in response to nitrogen step-down may be due to a synergistic effect of NtcA and other heterocyst-specific factor(s) or to the higher activity of NtcA exhibited in those differentiated cells (Ramasubramanian et al., 1994). If, as we imagine, there is little free iron in the heterocyst, FurA would be released from its own promoter, thereby allowing binding of NtcA, whose activity in the heterocyst could be enhanced by increased levels of 2-oxoglutarate (Herrero et al., 2001).
It has been shown that Anabaena sp. responds to nitrogen deprivation in several phases. First, if it was previously grown with ammonium, those of its genes repressed by ammonium ions are de-repressed (Cai & Wolk, 1997). Second, genes required for heterocyst differentiation, or related to the structure and function of heterocysts, are up-regulated in proheterocysts (Black et al., 1993; Wolk et al., 1993; Yoon & Golden, 2001). Finally, other genes related to heterocyst function are up-regulated, again not all at the same time (Ehira et al., 2003; Ehira & Ohmori, 2006). FurA, which seems to belong to the second group of genes, could play diverse roles in those differentiating cells. Although formation of the glycolipid layer of the heterocyst envelope presumably blocks the entry of ions directly from the medium, heterocysts continue to interact with vegetative cells, and so may have continued access to supplies of iron from vegetative cells. If developing heterocysts are deficient in free iron due to the large amount of iron required by the electron carriers that are involved in nitrogen fixation, it appears unlikely that FurA would serve only as a repressor in those cells.
In heterotrophic bacteria, under certain stress conditions, Fur proteins activate transcription of several genes involved in cell defence (Lee et al., 2004). Accordingly, because the nitrogenase components are highly sensitive to oxygen, FurA may coordinate the availability of iron with responses to potential oxidative stress. Early activation of FurA in the proheterocyst when the heterocyst envelope is not yet completely formed may support this idea. Because heterocysts are not completely anoxic, we hypothesize that the low pO2 in the heterocyst could activate the expression of enzymes that are involved in a concerted defence against oxidative stress. This hypothesis could receive support from the presence of catalase and superoxide dismutase in the heterocyst (Wolk et al., 1994; Li et al., 2002). Alternatively, FurA could exert a protective function in a heterocyst similar to that of DpsA (Peña & Bullerjahn, 1995). Although further studies have to be done to elucidate the role of FurA in the heterocyst, our results suggest that FurA is a principal node in the complex regulatory network that links iron and nitrogen-metabolic pathways and a key protein for the metabolism of the heterocyst.
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ACKNOWLEDGEMENTS |
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Edited by: K. Forchhammer
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REFERENCES |
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Andrews, S. C., Robinson, A. K. & Rodríguez-Quiñones, F. (2003). Bacterial iron homeostasis. FEMS Microbiol Rev 27, 215–237.[CrossRef][Medline]
Bagg, A. & Neilands, J. B. (1987). Ferric uptake regulation protein acts as a repressor, employing iron(II) as a cofactor to bind the operator of an iron transport operon in Escherichia coli. Biochemistry 26, 5471–5477.[CrossRef][Medline]
Bes, M. T., Hernández, J. A., Peleato, M. L. & Fillat, M. F. (2001). Cloning, overexpression and interaction of recombinant Fur from the cyanobacterium Anabaena PCC 7119 with isiB and its own promoter. FEMS Microbiol Lett 194, 187–192.[CrossRef][Medline]
Black, T. A., Cai, Y. & Wolk, C. P. (1993). Spatial expression and autoregulation of hetR, a gene involved in the control of heterocyst development in Anabaena. Mol Microbiol 9, 77–84.[Medline]
Böhme, H. & Haselkorn, R. (1988). Molecular cloning and nucleotide sequence analysis of the gene coding for heterocyst ferredoxin from the cyanobacterium Anabaena sp. strain PCC 7120. Mol Gen Genet 214, 278–285.[CrossRef][Medline]
Busby, S. & Ebright, R. H. (1997). Transcription activation at class II CAP-dependent promoters. Mol Microbiol 23, 853–859.[CrossRef][Medline]
Cai, Y. & Wolk, C. P. (1997). Nitrogen deprivation of Anabaena sp. strain PCC 7120 elicits rapid activation of a gene cluster that is essential for uptake and utilization of nitrate. J Bacteriol 179, 258–266.
Chastain, C. J., Brusca, J. S., Ramasubramanian, T. S., Wei, T. F. & Golden, J. W. (1990). A sequence-specific DNA-binding factor (VF1) from Anabaena sp. strain PCC 7120 vegetative cells binds to three adjacent sites in the xisA upstream region. J Bacteriol 172, 5044–5051.
Cheng, L. J., Shi, L., Lafiti, A. & Zhang, C.-C. (2006). A pair of iron-responsive genes encoding protein kinases with a Ser/Thr-kinase domain and a His-kinase domain are regulated by NtcA in the cyanobacterium Anabaena sp. strain PCC 7120. J Bacteriol 188, 4822–4829.
Cormack, B. P., Valdivia, R. H. & Falkow, S. (1996). FACS-optimized mutants of the green fluorescent protein (GFP). Gene 173, 33–38.[CrossRef][Medline]
De Lorenzo, V., Herrero, M., Giovannini, F. & Neilands, J. B. (1988). Fur (ferric uptake regulation) protein and CAP (catabolite-activator protein) modulate transcription of fur gene in Escherichia coli. Eur J Biochem 173, 537–546.[Medline]
Ehira, S. & Ohmori, M. (2006). NrrA, a nitrogen-responsive response regulator facilitates heterocyst development in the cyanobacterium Anabaena sp. strain PCC 7120. Mol Microbiol 59, 1692–1703.[CrossRef][Medline]
Ehira, S., Ohmori, M. & Sato, N. (2003). Genome-wide expression analysis of the responses to nitrogen deprivation in the heterocyst-forming cyanobacterium Anabaena sp. strain PCC 7120. DNA Res 10, 97–113.[Abstract]
Elhai, J. & Wolk, C. P. (1988a). A versatile class of positive-selection vectors based on the nonviability of palindrome-containing plasmids that allows cloning into long polylinkers. Gene 68, 119–138.[CrossRef][Medline]
Elhai, J. & Wolk, C. P. (1988b). Conjugal transfer of DNA to cyanobacteria. Methods Enzymol 167, 747–754.[Medline]
Falkowski, P. G., Barber, R. T. & Smetacek, V. V. (1998). Biogeochemical controls and feedbacks on ocean primary production. Science 281, 200–207.[CrossRef][Medline]
Fan, Q., Huang, G., Lechno-Yossef, S., Wolk, C. P., Kaneko, T. & Tabata, S. (2005). Clustered genes required for synthesis and deposition of envelope glycolipids in Anabaena sp. strain PCC 7120. Mol Microbiol 58, 227–243.[CrossRef][Medline]
Frías, J. E., Flores, E. & Herrero, A. (1994). Requirement of the regulatory protein NtcA for the expression of nitrogen assimilation and heterocyst development genes in the cyanobacterium Anabaena sp. PCC 7120. Mol Microbiol 14, 823–832.[Medline]
Frías, J. E., Flores, E. & Herrero, A. (2000). Activation of the Anabaena nir operon promoter requires both NtcA (CAP family) and NtcB (LysR family) transcription factors. Mol Microbiol 38, 613–625.[CrossRef][Medline]
Fuangthong, M., Herbig, A. F., Bsat, N. & Helmann, J. D. (2002). Regulation of the Bacillus subtilis fur and perR genes by PerR: not all members of the PerR regulon are peroxide inducible. J Bacteriol 184, 3276–3286.
Hernández, J. A., López-Gomollón, S., Bes, M. T., Fillat, M. F. & Peleato, M. L. (2004a). Three fur homologues from Anabaena sp. PCC7120: exploring reciprocal protein-promoter recognition. FEMS Microbiol Lett 236, 275–282.[CrossRef][Medline]
Hernández, J. A., Peleato, M. L., Fillat, M. F. & Bes, M. T. (2004b). Heme binds to and inhibits the DNA-binding activity of the global regulator FurA from Anabaena sp. PCC 7120. FEBS Lett 577, 35–41.[CrossRef][Medline]
Hernández, J. A., Muro-Pastor, A. M., Flores, E., Bes, M. T., Peleato, M. L. & Fillat, M. F. (2006a). Identification of a furA cis antisense RNA in the cyanobacterium Anabaena sp. PCC 7120. J Mol Biol 355, 325–334.[CrossRef][Medline]
Hernández, J. A., López-Gomollón, S., Muro-Pastor, A., Valladares, A., Bes, M. T., Peleato, M. L. & Fillat, M. F. (2006b). Interaction of FurA from Anabaena sp. PCC 7120 with DNA: a reducing environment and the presence of Mn2+ are positive effectors in the binding to isiB and furA promoters. Biometals 19, 259–268.[CrossRef][Medline]
Herrero, A., Muro-Pastor, A. M. & Flores, E. (2001). Nitrogen control in cyanobacteria. J Bacteriol 183, 411–425.
Jiang, F., Mannervik, B. & Bergman, B. (1997). Evidence for redox regulation of the transcription factor NtcA, acting both as an activator and a repressor, in the cyanobacterium Anabaena PCC 7120. Biochem J 327, 513–517.
Kolb, A., Busby, S., Buc, H., Garges, S. & Adhya, S. (1993). Transcriptional regulation by cAMP and its receptor protein. Annu Rev Biochem 62, 749–795.[CrossRef][Medline]
Kustka, A., Carpenter, E. J. & Sanudo-Wilhelmy, S. A. (2002). Iron and marine nitrogen fixation: progress and future directions. Res Microbiol 153, 255–262.[Medline]
Laurent, S., Chen, H., Bédu, S., Ziarelli, F., Peng, L. & Zhang, C. C. (2005). Nonmetabolizable analogue of 2-oxoglutarate elicits heterocyst differentiation under repressive conditions in Anabaena sp. PCC 7120. Proc Natl Acad Sci U S A 102, 9907–9912.
Lee, H. W., Choe, Y. H., Kim, D. K., Jung, S. Y. & Lee, N. G. (2004). Proteomic analysis of a ferric uptake regulator mutant of Helicobacter pylori: regulation of Helicobacter pylori gene expression by ferric uptake regulator and iron. Proteomics 4, 2014–2027.[CrossRef][Medline]
Li, T., Huang, X., Zhou, R., Liu, Y., Li, B., Nomura, C. & Zhao, J. (2002). Differential expression and localization of Mn and Fe superoxide dismutases in the heterocystous cyanobacterium Anabaena sp. strain PCC 7120. J Bacteriol 184, 5096–5103.
Liang, J., Scappino, L. & Haselkorn, R. (1993). The patB gene product, required for growth of the cyanobacterium Anabaena sp. strain PCC 7120 under nitrogen-limiting conditions, contains ferredoxin and helix-turn-helix domains. J Bacteriol 175, 1697–1704.
Luque, I., Zabulon, G., Contreras, A. & Houmard, J. (2001). Convergence of two global transcriptional regulators on nitrogen induction of the stress-acclimation gene nblA in the cyanobacterium Synechococcus sp. PCC 7942. Mol Microbiol 41, 937–947.[CrossRef][Medline]
Muro-Pastor, A. M., Valladares, A., Flores, E. & Herrero, A. (1999). The hetC gene is a direct target of the NtcA transcriptional regulator in cyanobacterial heterocyst development. J Bacteriol 181, 6664–6669.
Muro-Pastor, M. I., Reyes, J. C. & Florencio, F. J. (2001). Cyanobacteria perceive nitrogen status by sensing intracellular 2-oxoglutarate levels. J Biol Chem 276, 38320–38328.
Peña, M. M. & Bullerjahn, G. S. (1995). The DpsA protein of Synechococcus sp. strain PCC7942 is a DNA-binding hemoprotein. Linkage of the Dps and bacterioferritin protein families. J Biol Chem 270, 22478–22482.
Ramasubramanian, T. S., Wei, T. F. & Golden, J. W. (1994). Two Anabaena sp. strain PCC 7120 DNA-binding factors interact with vegetative cell- and heterocyst-specific genes. J Bacteriol 176, 1214–1223.
Rippka, R. (1988). Isolation and purification of cyanobacteria. Methods Enzymol 167, 3–27.[Medline]
Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989). Molecular Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
Straus, N. A. (1994). Iron deprivation: physiology and gene regulation. In The Molecular Biology of Cyanobacteria, pp. 731–750. Edited by D. A. Bryant. Dordrecht: Kluwer Academic Publishers.
Valladares, A., Muro-Pastor, A. M., Fillat, M. F., Herrero, A. & Flores, E. (1999). Constitutive and nitrogen-regulated promoters of the petH gene encoding ferredoxin : NADP+ reductase in the heterocyst-forming cyanobacterium Anabaena sp. FEBS Lett 449, 159–164.[CrossRef][Medline]
Wolk, C. P., Elhai, J., Kuritz, T. & Holland, D. (1993). Amplified expression of a transcriptional pattern formed during development of Anabaena. Mol Microbiol 7, 441–445.[Medline]
Wolk, C. P., Ernst, A. & Elhai, J. (1994). Heterocyst metabolism and development. In The Molecular Biology of Cyanobacteria, pp. 769–823. Edited by D. A. Bryant. Dordrecht: Kluwer Academic Publishers.
Yoon, H. S. & Golden, J. W. (2001). PatS and products of nitrogen fixation control heterocyst pattern. J Bacteriol 183, 2605–2613.
Zhang, Z., Gosset, G., Barabote, R., Gonzalez, C. S., Cuevas, W. A. & Saier, M. H., Jr (2005). Functional interactions between the carbon and iron utilization regulators, Crp and Fur, in Escherichia coli. J Bacteriol 187, 980–990.
Zheng, M., Doan, B., Schneider, T. D. & Storz, G. (1999). OxyR and SoxRS regulation of fur. J Bacteriol 181, 4639–4643.
Received 19 July 2006;
revised 15 September 2006;
accepted 18 September 2006.
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