Cellular differentiation and the NtcA transcription factor in filamentous cyanobacteria

Authors

  • Antonia Herrero,

    Corresponding author
    1. Instituto de Bioquímica Vegetal y Fotosíntesis, Consejo Superior de Investigaciones Científicas, Centro de Investigaciones Científicas Isla de la Cartuja, Universidad de Sevilla, Avda. Américo Vespucio s/n, E-41092 Seville, Spain
      *Corresponding author. Tel.: +34-95-448-9522; fax: +34-95-446-0065, E-mail address: herrero@ibvf.csic.es
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  • Alicia M. Muro-Pastor,

    1. Instituto de Bioquímica Vegetal y Fotosíntesis, Consejo Superior de Investigaciones Científicas, Centro de Investigaciones Científicas Isla de la Cartuja, Universidad de Sevilla, Avda. Américo Vespucio s/n, E-41092 Seville, Spain
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  • Ana Valladares,

    1. Instituto de Bioquímica Vegetal y Fotosíntesis, Consejo Superior de Investigaciones Científicas, Centro de Investigaciones Científicas Isla de la Cartuja, Universidad de Sevilla, Avda. Américo Vespucio s/n, E-41092 Seville, Spain
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  • Enrique Flores

    1. Instituto de Bioquímica Vegetal y Fotosíntesis, Consejo Superior de Investigaciones Científicas, Centro de Investigaciones Científicas Isla de la Cartuja, Universidad de Sevilla, Avda. Américo Vespucio s/n, E-41092 Seville, Spain
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*Corresponding author. Tel.: +34-95-448-9522; fax: +34-95-446-0065, E-mail address: herrero@ibvf.csic.es

Abstract

Some filamentous cyanobacteria can undergo a variety of cellular differentiation processes that permit their better adaptation to certain environmental conditions. These processes include the differentiation of hormogonia, short filaments aimed at the dispersal of the organism in the environment, of akinetes, cells resistant to various stress conditions, and of heterocysts, cells specialized in the fixation of atmospheric nitrogen in oxic environments. NtcA is a transcriptional regulator that operates global nitrogen control in cyanobacteria by activating (and in some cases repressing) many genes involved in nitrogen assimilation. NtcA is required for the triggering of heterocyst differentiation and for subsequent steps of its development and function. This requirement is based on the role of NtcA as an activator of the expression of hetR and other multiple genes at specific steps of the differentiation process. The products of these genes effect development as well as the distinct metabolism of the mature heterocyst. The different features found in the NtcA-dependent promoters, together with the cellular level of active NtcA protein, should have a role in the determination of the hierarchy of gene activation during the process of heterocyst differentiation.

1Introduction

Cyanobacteria are ancient organisms that by having developed oxygenic photosynthesis, leading to the accumulation of oxygen in the atmosphere, have played a crucial role in the evolution of our planet. Together with chloroplasts, cyanobacteria constitute a coherent phylogenetic group of high rank in the Bacteria domain of life [1]. Currently, cyanobacteria have a wide ecological distribution and contribute an important fraction of the primary productivity of the oceans, in which the input of fixed nitrogen due to cyanobacterial diazotrophy is highly relevant [2].

Cyanobacteria display a relatively wide range of morphological diversity, that ranges from unicellular, rod- or coccus-shaped, in some cases grouped into defined aggregates, to filamentous forms exhibiting different degrees of filament complexity [3]. Despite this morphological diversity, cyanobacteria are rather homogeneous in their metabolic way of living, which is primarily based on photoautotrophy with CO2 fixation through the reductive pentose phosphate pathway.

With regard to the assimilation of nitrogen, cyanobacteria preferentially use inorganic nitrogen for growth. Nitrate and ammonium are excellent sources of nitrogen for cyanobacteria in general, and many representatives, both unicellular and filamentous, are also able to perform the fixation of atmospheric nitrogen. Some simple organic molecules such as urea can also be efficiently used by a number of cyanobacteria, some of which are also able to grow at the expenses of some amino acids, such as arginine or glutamine, or nitrogen-containing bases (for a review, see [4]). In recent years, a good deal of knowledge has accumulated on the molecular details of the pathways for the assimilation of nitrogen by cyanobacteria, including information on a number of transport systems for the uptake of nitrogen nutrients, and numerous genetic systems encoding elements of those pathways have been identified and characterized (see [5]). Whatever the environmental nitrogen source, its intracellular processing renders ammonium, which is assimilated mainly through the glutamine synthetase/glutamate synthase pathway, thus providing the basis for a coordinated regulation of nitrogen assimilation.

Global regulation of nitrogen assimilation exerted through ammonium-promoted repression of genes involved in the assimilation of alternative nitrogen sources is a common theme in bacteria, and is also operative in cyanobacteria. Nevertheless, the molecular mechanism by which nitrogen control is exerted in the cyanobacteria is distinctive, with the transcriptional regulator NtcA playing a central role in it.

2Nitrogen control and the NtcA transcription factor

Many microorganisms that are capable of assimilating a variety of nitrogen sources exhibit a preference for ammonium (or, in some cases, glutamine) over other compounds which are therefore considered as alternative nitrogen sources. This preference of assimilation is sustained by a regulatory phenomenon termed nitrogen control (N control) that ensures that permeases and enzymes of the assimilatory pathways for alternative nitrogen sources are not expressed when the cells are exposed to a non-limiting concentration of ammonium. N control also affects the genes amt (or amtB), encoding the ammonium permease, and glnA, encoding glutamine synthetase, whose high-level expression is essential for an efficient assimilation of ammonium when it is present at a low concentration in the extracellular medium. At least five different molecular mechanisms operating N control have been identified in bacteria, those of the enterobacteria (also present with some variations in some other proteobacteria), Bacillus subtilis, Corynebacterium glutamicum, Methanococcus maripaludis, and the cyanobacteria.

2.1Nitrogen control in enterobacteria and some other bacteria

The best characterized bacterial N-control system is the one found in the enterics, whose core elements are the NtrB–NtrC two-component regulatory system (in which NtrB is the sensor and NtrC the response regulator), the PII-type signal transduction protein GlnB, and the glnD gene product uridylyltransferase, an enzyme that modifies or demodifies GlnB in response to, respectively, low and high cellular levels of glutamine (for recent reviews, see [6,7]). Activity of GlnB is not only affected by its uridylylation state but also by binding of 2-oxoglutarate (and ATP). Under nitrogen limitation, when GlnB is uridylylated and carries bound 2-oxoglutarate, NtrB phosphorylates NtrC producing NtrC-P that activates transcription of glnA and other genes. In contrast, under nitrogen excess, non-modified GlnB accumulates and interacts with NtrB stimulating its NtrC-P phosphatase activity, leading to accumulation of non-phosphorylated NtrC and thus to decreased expression of the NtrC-dependent genes. GlnB also affects the modification of glutamine synthetase by adenylylation, which negatively influences glutamine synthetase activity and increases its sensitivity to feed-back inhibitors. The glutamine synthetase adenylyltransferase (glnE gene product) is, as is also the case with the PII uridylyltransferase, sensitive to glutamine [6,7]. It is now known that a second PII-type protein, GlnK, is widely distributed in bacteria [7]. In its non-uridylylated state, GlnK appears to be an inhibitor of the ammonium permease [8]. Another relevant role of a GlnK protein has been described concerning the NifL–NifA regulatory system of nif gene expression in Azotobacter vinelandii[9], in which the inhibitory activity of NifL on NifA is stimulated by non-uridylylated PII (GlnK).

In the low-G + C content bacterium B. subtilis, N-regulated gene expression is controlled by the MerR-family proteins TnrA and GlnR [10]. Under N excess, GlnR represses glnA and other genes, and under N limitation TnrA activates the expression of genes like amtB-glnK and represses others like glnA. Glutamine synthetase is not subjected to post-translational modification in B. subtilis, but it is the target of feedback regulation mainly by glutamine. It has recently been shown that the feedback-inhibited form of glutamine synthetase directly interacts with TnrA blocking the DNA binding activity of this transcriptional regulator [11]. In the high-G + C content bacterium C. glutamicum N control is mediated by AmtR, a DNA-binding protein of the TetR/ArcR family that acts as a repressor of amt and other N-controlled genes [12]. One of the operons repressed by AmtR is amtB-glnK-glnD, and GlnK and GlnD are necessary for expression of N-controlled genes. It has been suggested that GlnK-UMP interacts with AmtR to release repression, and that in this bacterium N control responds to metabolic signal(s) other than glutamine [13]. Recently, in the methanogenic archaeon M. maripaludis, the NrpR protein has been identified. NrpR represses glnA and nif genes by binding to their promoters in cells exposed to ammonium, and represents a novel type of transcriptional regulator [14].

Perception of the C and N metabolic status of the cell is unknown in the case of C. glutamicum or M. maripaludis, but it appears to involve glutamine (sensed by glutamine synthetase) in B. subtilis, and glutamine (sensed by uridylyltransferase) and 2-oxoglutarate (sensed by GlnB or GlnK) in the enterobacteria.

2.2Nitrogen control in cyanobacteria

In cyanobacteria, a distinct N control mechanism has been identified. Ammonium is a preferred nitrogen source in these organisms, and its presence in the growth medium determines repression of genes encoding elements of the assimilation pathways for the alternative nitrogen sources N2, nitrate or urea (for a review, see [4]). In the absence of ammonium, NtcA, a transcriptional regulator of the CAP family, promotes expression of alternative nitrogen-source assimilation genes, such as those in the nir (nitrate assimilation) and urt (urea transport) operons, and of ammonium assimilation genes like amt and glnA, but can also act as a repressor of some genes [5]. The NtcA binding site in DNA has the sequence signature GTAN8TAC [5,15] of which the GTN10AC subset can be considered essential for binding [16]. In the NtcA-activated promoters, the NtcA-binding site is frequently centered at about −41.5 nucleotides with respect to the transcription start site (tsp), and these promoters also carry a −10 box in the form TAN3T. This promoter structure is similar to that of the Class II promoters activated by CAP [15], but NtcA-activated promoters in which an NtcA-binding site is found further upstream from the −41.5 position have moreover been identified (see below). Also, NtcA could bind at more than one site in the regulated promoter to effect regulation of gene expression. This is the case, for instance, of the nir operon promoter in Synechococcus sp. strain PCC 7942, in which an NtcA-binding site centered at −109.5 is found in addition to the one centered at −40.5 [15]. NtcA-repressor sites are located in positions overlapping the −35 or −10 promoter boxes or the transcription start site [5].

Details of NtcA action have been worked out mainly in the unicellular, non-nitrogen fixing cyanobacterium Synechococcus elongatus strain PCC 7942. The ntcA gene is autoregulatory showing a low level of expression in ammonium-grown cells and an increased expression, which is dependent on NtcA itself, in the absence of ammonium [15]. Additionally, over-expression of NtcA in S. elongatus does not override the need for ammonium deprivation to allow expression of N-regulated genes [17]. The transcriptional activity of NtcA appears therefore to be subjected to regulation so that NtcA becomes active when the cells perceive limitation of ammonium. NtcA binding in vitro to the S. elongatus glnA promoter [18], as well as in vitro activation of transcription at the glnA and ntcA promoters [19], is stimulated by 2-oxoglutarate. This metabolite has also been shown to stimulate expression of NtcA-dependent nitrogen assimilation genes in S. elongatus transformed with a gene encoding a 2-oxoglutarate permease [20]. Additionally, expression of the NtcA-dependent gene amt1 has been shown to be influenced not only by the nitrogen but also by the carbon supply of the cells [21]. All these observations point to 2-oxoglutarate as a key element in the C to N balance signaling pathway of S. elongatus. Consistently with its putative signaling role, determinations of 2-oxoglutarate levels in different cyanobacteria incubated under different conditions of nitrogen supply have indicated accumulation of 2-oxoglutarate under N-limiting conditions [22–25]. Because cyanobacteria lack 2-oxoglutarate dehydrogenase [26], the main metabolic role of 2-oxoglutarate in these organisms is incorporation of nitrogen through the glutamine synthetase-glutamate synthase cycle [27], which positions 2-oxoglutarate at the link of C and N metabolisms.

A PII-type protein, GlnB, which acts as a 2-oxoglutarate sensor, is present in cyanobacteria, although in these organisms GlnB is subjected to modification by phosphorylation rather than by uridylylation (for a review, see [28]). In S. elongatus, GlnB mediates the ammonium-promoted inhibition of nitrate uptake [29,30] and is required for high-level expression of NtcA-dependent genes under N deprivation [31,32]. Although the mechanism behind this relationship between NtcA and GlnB is not yet known, regulation by these two 2-oxoglutarate-responsive proteins may have synergistic effects. In Nostoc punctiforme strain ATCC 29133, inability to segregate a knockout mutant of the glnB gene has led to the suggestion that it may have an essential function in heterocyst-forming cyanobacteria [33]. On the other hand, no role for glutamine as a putative effector in N control has been found in cyanobacteria. Glutamine synthetase is not subjected to adenylylation in these organisms, but it can be the target of feedback inhibition by some amino acids (Asp, Ala, Ser, Gly) and nucleotides (AMP, ADP) (reviewed in [4]). Additionally, in Synechocystis sp. strain PCC 6803, glutamine synthetase activity has been shown to be negatively regulated by binding of two inhibitory factors, the gifA and gifB gene products, whose cellular levels are determined by NtcA-dependent repression, which takes place in the absence of ammonium [34,35]. Thus, in contrast to the situation in the enterics and Gram-positive bacteria in which glutamine plays a key role in N control, 2-oxoglutarate, influencing the activity of NtcA and GlnB, is emerging as the key metabolite in the regulation of nitrogen assimilation in cyanobacteria.

3Cellular differentiation in filamentous cyanobacteria

Many filamentous cyanobacteria can undergo one or several of a variety of cellular differentiation processes that most commonly take place as adaptive responses to environmental changes. In general, these differentiation processes allow the cyanobacterium to make use of some nutritional options or to better stand unfavourable conditions, but are dispensable for the survival of the organism under other circumstances. In some cases, multiple relationships, both nutritional and regulatory, are established between the different types of cells of the filament, so that in some respects the filamentous cyanobacteria can be regarded as simple multicellular organisms.

The remainder of this review will be devoted to cellular differentiation processes widely studied in representatives of the order Nostocales, but a peculiar type of differentiated cells has recently been identified in non-heterocystous marine cyanobacteria of the genus Thrichodesmium. Members of this genus make a significant contribution to global N2 fixation in the oceans and are able to fix N2 in the light under oxic conditions. In these cyanobacteria, nitrogenase is located in specialized cells called diazocytes that form short stretches in the trichome [36,37]. The differentiation of diazocytes will undoubtedly be the subject of detailed study in the near future.

3.1Differentiation of hormogonia

Hormogonia are short, motile filaments of small cells, generally distinguishable both in morphology and shape from the mature trichome (Fig. 1), that function in the dispersal of the cyanobacterium in the environment. The differentiation of hormogonia takes place through a number of rapid cell division events that are not coupled to net DNA synthesis or to an increase in cell biomass, but produces partitioning of the many copies of the chromosome that are usually present in vegetative cyanobacterial cells [38,39]. The ftsZ gene (which in Escherichia coli has been shown to encode a self-assembling, filament-forming protein essential for cell division) has been cloned from the hormogonium-forming cyanobacterium Fremyella diplosiphon (Calothix sp. strain PCC 7601) and characterized [40]. ftsZ has been shown to increase its expression preceding the peak of cell division, after a shift to conditions that induce hormogonium formation. This observation suggests that, as seems to be also the case in E. coli, the amount of FtsZ protein could be rate-limiting for cell division in F. diplosiphon[40], at least during the burst of cell division that produces the hormogonium. Hormogonia represent a transient state of the cyanobacterium that, subsequently, losses motility and resumes the synthesis of macromolecules leading to the production of mature, vegetative trichomes.

Figure 1.

Cyanobacterial filaments showing vegetative and differentiated cells. Left, Nostoc sp. strain PCC 9203 showing hormogonia (HO); upper right, Anabaena cylindrica (ATCC 29414) showing akinetes (A) and heterocysts (H); lower right, strain 9v, a natural isolate from Mkindo (Tanzania) showing intercalary and terminal heterocysts (H). The photographs show filaments from cultures grown in BG110 medium (combined nitrogen-free medium [3]).

In some strains of the genera Nostoc, Tolypothrix and Calothrix, the differentiation of hormogonia may take place as a transient stage of the cell cycle (see [41]), and in the case of symbiotic associations with other organisms, hormogonia represent the infective form of the cyanobacterium that initiates the contact with the partner, hormogonium development being influenced both positively and negatively by host-released factors during the progression of the symbiosis [42,43]. Nevertheless, in many cyanobacteria the differentiation of hormogonia seems to be a dispensable event taking place in response to changes in diverse external factors, including light and nutrients, that in fact can affect the differentiation process either positively or negatively (see [41]). It can be envisioned that rather than in response to a specific environmental cue, the differentiation of hormogonia may respond to changes that could impact the coordination between cell growth and division. In particular, the relation of hormogonium differentiation to nitrogen availability is apt to be nonspecific, and in fact hormogonium differentiation can be induced both in the presence and absence of combined nitrogen (see e.g. [44] for hormogonia development in nitrate-containing medium). However, a mutant of the global nitrogen regulator NtcA derived from N. punctiforme has been reported to differentiate hormogonia at lower frequency than the wild-type strain when tested in co-culture with its symbiotic partner Anthoceros punctatus, and is unable to infect it [45]. In contrast, N. punctiforme strains with mutations in hetR or hetF genes (involved in heterocyst development, see below) infect A. punctatus at frequencies similar to that of the wild type [45].

3.2Differentiation of akinetes

Akinetes are cells distinguishable from vegetative cells of the filament by their larger size, thicker cell wall and conspicuous granulation (Fig. 1) consisting of cyanophycin and glycogen. Akinetes are considered as propagating, or perennating, bodies exhibiting resistance to adverse conditions, mainly cold and dessication. However, similarly to Azotobacter cysts, akinetes are sensitive to high temperatures, in this respect differing from bacterial endospores [46]. Under favourable conditions, akinetes germinate producing short filaments that emerge through ruptures of the akinete cell wall (see e.g. [47]). The amount of DNA is generally reported to be similar in akinetes and vegetative cells, and while some metabolic activities such as CO2 fixation are very low in akinetes, the rate of respiration is often high (see [48]). Also, akinetes have been shown to make at least a few proteins, so that they seem to maintain some, although low, metabolic activity [49].

Similar to the situation with the development of hormogonia, no single environmental trigger has been demonstrated to promote akinete development. Under laboratory conditions, akinetes are profusedly formed at the end of the exponential growth phase, their appearance being delayed by factors that prolong active growth of cultures, so that the most widely recognized factors influencing akinete differentiation, such as light or phosphate limitation, could act by causing energy limitation [46]. Akinete germination can be induced by dilution of stationary-phase cultures into fresh medium, and in general by changes favouring active growth of cultures, and it should be aided by their usually high nitrogen (cyanophycin) and carbon (glycogen) reserves content. Initiation of akinete germination does not require DNA synthesis, but may be sustained by cell division events distributing between the newly formed vegetative cells the various copies of the chromosome present in the akinete. In this context, it is worth mentioning the report [50] that mutation of genes ftn, which would encode products containing a DnaJ motif, causes the formation of akinete-like cells in Anabaena sp. strain PCC 7120 (also known as Nostoc sp. strain PCC 7120), a strain not previously recognized as capable of akinete differentiation. This observation suggests that these genes could be involved in akinete differentiation through an effect on cell division.

In some cyanobacteria, in the absence of combined nitrogen akinetes are frequently formed adjacent to a heterocyst, which specifically differentiates in response to combined nitrogen deprivation (see below), and the addition of some nitrogen nutrients like nitrate or urea inhibits akinete formation (e.g. [51]). Nevertheless, in other strains akinetes first appear distant from heterocysts in the absence of combined nitrogen, and can differentiate in the presence of combined nitrogen and thus in the absence of heterocysts [52]. Information currently available suggests to us that limitation of nitrogen may be a factor that induces akinete development indirectly by provoking a decrease of the growth rate.

The differentiation of akinetes has evident connections to the differentiation of heterocysts. In some strains the pattern of heterocyst distribution determines that of akinete distribution in the absence of combined nitrogen [46]. Additionally, some common specific structural components have been identified in the wall of both cell types that are synthesized during the differentiation processes (see [53]), and mutation in Anabaena variabilis (strain ATCC 29413) of the gene hepA (which in Anabaena sp. strain PCC 7120 has been characterized as involved in formation of the heterocyst polysaccharide layer) also impairs akinete development [53]. In Nostoc ellipsosporum, inactivation of the gene hetR (whose mutation prevents heterocyst differentiation, see below) has been reported to impair also akinete differentiation, and the hetR gene is expressed also in the akinetes [54]. It has been suggested that the differentiation of heterocysts may have evolved based on that of akinetes, which would have existed formerly [53], with some genetic elements (e.g. hetR and hepA) acting in a supposedly common stem of the differentiation of both types of cells, while other elements (e.g. hetP) would act later and be specific for the differentiation of heterocysts [54]. On the other hand, in N. punctiforme, inactivation of hetR has been reported to prevent heterocyst development but permit the formation of akinete-like cells [45]. Strain differences might respond for these apparently contrasting results. Alternatively, akinete-like cells could develop in the N. ellipsosporum hetR mutant as cells more resistant to certain stress conditions, but more similar in morphology to vegetative cells than to akinetes of the wild type and, thus, could have gone unnoticed (see [45]). If this were the case, hetR would have a role in akinete differentiation but, in contrast to heterocyst differentiation (see below), would not be required to trigger the process.

Research on akinete differentiation may experience a revival thanks to the recent identification of AvaK, a protein that may serve as a marker for the process [55].

3.3Differentiation of heterocysts

Heterocysts are cells highly specialized in the fixation of atmospheric nitrogen under oxic conditions that some filamentous cyanobacteria differentiate when combined nitrogen becomes limiting (Fig. 1). Heterocysts are terminally differentiated cells that neither divide, consistent with the lack of the FtsZ protein in these cells [56], nor, after a certain point in the developmental process, revert to the vegetative cell state. Heterocyst death causes, in the case of intercalary heterocysts, breakage of the filament at the point occupied by the moribund heterocyst.

Heterocysts exhibit conspicuous differences, both in structure and function, with the vegetative cells from which they originate. These differences are aimed at the expression of the nitrogen fixation machinery, at increasing the efficiency of the nitrogen fixation reaction, and at protection of the nitrogen fixation machinery against oxygen. The differential traits of the heterocyst include the presence of supplemental glycolipid and polysaccharide layers in the cell envelope, aimed at hampering the influx of gases; lack of activity of the photosystem II, avoiding photosynthetic oxygen production; increased respiration, eliminating free oxygen and also contributing to the provision of energy for the nitrogen fixation reaction; and lack of photosynthetic CO2 fixation, thus avoiding distracting energy and reducing power to processes other than nitrogen fixation (for a detailed review see [53]). During the process of heterocyst differentiation, several steps have traditionally been distinguished based mainly on physiological and ultrastructural evidence (see e.g. [57]).

When considering the differentiation of “first generation” heterocysts, i.e. differentiation triggered by exhaustion of sources of combined nitrogen, the first event is perception of nitrogen stress. This leads to an increase in general proteolysis and, in particular, to degradation of the phycobiliproteins, photosynthetic accessory pigments that may account for up to 50% of the cellular protein, thus producing the first microscopic sign of differentiation as a deficiency in fluorescence of the cells that start the route of development. Progression of differentiation produces the so-called proheterocysts, an intermediate stage that differs in shape and granulity from vegetative cells [53]. Proheterocysts undergo a series of traceable morphological changes (see [57,58]) that leads to the formation of the heterocyst-specific envelope and reorganization of intracellular membranes, more or less concomitant with characteristic changes in cell metabolism such as an increase in respiration and, finally, expression of nitrogenase activity, that in some cyanobacteria is preceded by several genomic reorganizations effected through site-specific recombinational events [53]. Based on the fact that certain mutants unable to form a proper heterocyst envelope are also unable to complete protoplast maturation, it has been suggested that the establishment of the barrier to oxygen might constitute a developmental checkpoint that could trigger the process of maturation [59,60].

In the diazotrophic filament of cyanobacteria, vegetative cells and heterocysts are mutually interdependent relying on metabolite exchanges that take place between the two types of cells. The heterocysts provide fixed nitrogen throughout the filament. Ammonium resulting from the reduction of N2 is incorporated inside the heterocyst into glutamate through the action of glutamine synthetase, whose activity is high in these cells, to render glutamine. It has been suggested that glutamine is the N-containing metabolite that is exported out of the heterocysts and made available to the vegetative cells [61,62], but the possibility that some other amino acids are also transferred should be considered. Although the mechanism of transference of fixed nitrogen in the diazotrophic filament is unknown, the involvement of uptake amino acid permeases in diazotrophic growth of Anabaena sp. strain PCC 7120 has suggested the hypothesis that amino acids can be exported from the heterocysts to the periplasmic space, which is continuous along the filament, from where they could be taken up by the vegetative cells through amino acid permeases [63]. On the other hand, although this point has been the subject of some controversy (see [53]), it appears that heterocysts are deficient in glutamate synthase [62,64], implying that glutamate has to be transferred to the heterocysts from the vegetative cells and/or synthesized in the heterocysts by a pathway alternative to that of glutamate synthase.

Since heterocysts have lost the capacity of photosynthetic CO2 fixation, the activity of nitrogen fixation in these cells depends upon the supply by the adjacent vegetative cells of reduced carbon compounds to be used as sources of reductant and of the substrate necessary for the incorporation of the ammonium derived from N2 reduction. Sucrose is considered a likely candidate for reduced carbon vehicle [53,65].

The distinctive morphological and physiological traits of the heterocysts are the consequence of a differential program of gene expression relative to that operating in the vegetative cells. Thus, a number of genes, such as those encoding the enzyme nitrogenase, are expressed only in the mature heterocyst or, such as the devBCA genes (see below), preferentially during the intermediate stages of heterocyst development, whereas other sets of genes, e.g. those encoding ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), are actively expressed in the vegetative cells but not in the heterocyst. Some genes can still be expressed in both types of cells, as is the case of glnA, encoding glutamine synthetase. As we will describe later in more detail, in Anabaena sp. strain PCC 7120 this and other genes can be transcribed from the same promoter in heterocysts and, at least under some conditions, in vegetative cells.

In the remainder of this review we will analyze present knowledge about the events of activation of gene expression that trigger the initiation of heterocyst differentiation, as well as those that take place during the intermediate stages of heterocyst differentiation and in the mature heterocyst, focusing on the function of the global nitrogen regulator NtcA that appears to play a crucial role at regulation of gene expression throughout the whole developmental process. Table 1 summarizes some genes that have been identified as involved in heterocyst differentiation or function. Some of these genes will be considered below, but for a more comprehensive description of them, as well as for discussion of possible mechanisms responsible for the establishment of the pattern of distribution of heterocysts along the filament, the reader is referred to some other recent reviews [39,42,95,121].

Table 1.  Some genes involved in heterocyst development or function
GeneProduct homology or (putative) functionReference(s)
  1. All genes first identified in Anabaena sp. strain PCC 7120 except those identified in N. punctiforme (ATCC 29133) (a), Anabaena sp. strain PCC 7119 (b), Nostoc commune (c), N. ellipsosporum (d) or A. variabilis (ATCC 29413) (e).

Early events
ntcANitrogen regulation; autoregulatory gene[66,67]
patAPattern formation[68]
patSIntercellular inhibition of heterocyst formation[69,70]
hanASimilar to E. coli protein HU[71]
hetRAutoregulatory gene; autoproteolytic and DNA-binding activities[72–75]
hetCABC-type exporter[76]
hetPUnknown function[77]
hetLNon essential positive-acting element[78]
hetFaRequired for localization of HetR in differentiating heterocysts[79]
   
Maturation
devHPutative DNA binding protein[80]
hetNPattern maintenance[81–83]
hglB(hetM), hglC, hglD, hglEaStructural genes for glycolipid biosynthesis[81,82,84]
hglK, devBCATransport and deposition of heterocyst envelope glycolipids[59,85]
hepK, devRaTwo-component regulatory system; heterocyst polysaccharide biosynthesis[86–88]
hepC, hepAHeterocyst envelope polysaccharide biosynthesis[87,89]
abp2 abp3DNA-binding proteins required for expression of hepC and hepA[90]
hcwAAutolysin required for heterocyst maturation[60]
pbpBPutative penicillin-binding protein[91]
rfbPUndecaprenyl-phosphate galactosephosphotransferase[92]
pknDProtein kinase[93]
prpA, pknEProtein phosphatase and kinase, respectively[94]
xisA, xisC, xisFExcisases involved in DNA rearrangementsReviewed in [95]
xisH, xisIExcision of the fdxN DNA-intervening element[96]
patBPutative helix-turn-helix and ferredoxin domains[97,98]
   
Function
zwfa, opcAaGlucose-6-phosphate dehydrogenase and allosteric effector[99,100]
cox2, cox3 operonsTerminal respiratory oxidases[101,102]
petHbFerredoxin-NADP+ reductase[103,104]
fdxHFerredoxin[105,106]
fdxNBacterial-type ferredoxin[107,108]
nif genesNitrogenase structural and maturation genes[109,110]
hupLSUptake hydrogenase[111–113]
glbNcCyanoglobin[114,115]
glnAGlutamine syntethase[66,116]
argLdArginine biosynthesis[117]
cphAe, cphBCyanophycin synthetase and cyanophycinase[118,119]
idhIsocitrate dehydrogenase[64,120]

4Role of NtcA at the initiation of heterocyst differentiation

Heterocyst differentiation takes place upon exhaustion of combined nitrogen and, as mentioned above, involves a number of changes, both at the structural and metabolic levels, that turn cells into efficient nitrogen fixation factories. Although different heterocyst distribution patterns can be found in different filamentous cyanobacteria, the subject of heterocyst differentiation and distribution has been studied at the molecular level almost exclusively in the Nostocaceae, in which heterocysts appear at semiregular intervals along the filament with a frequency of one heterocyst every ca. 10–15 vegetative cells.

A number of genes has been identified as involved in heterocyst differentiation and/or function. These genes can be ascribed to different stages of the process of heterocyst differentiation on the basis of the phenotypes observed upon their mutation or overexpression. Genes whose products are involved in early steps of differentiation are referred to as early genes, whereas those whose products are involved in maturation until the formation of a functional heterocyst are referred to as late genes. In the case of some genes whose products exert a positive action at the initiation of the process, mutation leads to absence of any sign of differentiation, whereas mutation of some other early genes still permits the observation of some initial symptoms of differentiation, such as a pattern of spaced nonfluorescent cells. In the case of genes whose products exert a negative action, mutation can lead to a high frequency, and eventually the formation of clusters, of heterocysts (multiple contiguous heterocysts phenotype, Mch). The same phenotype can be produced by overexpression of some positive-acting genes. If the gene product acts at later stages, the phenotype of the corresponding knockout mutant is less severe, so that heterocysts are observed but they are morphologically aberrant or simply not functional. It should be noted, however, that the time at which a certain gene is induced can be considerably earlier than that at which its product exerts a discernible effect during the course of the differentiation process.

In those strains in which the subject has been studied, Anabaena sp. strain PCC 7120 [66,67], A. variabilis[122], and N. punctiforme[45], NtcA has been shown to be required for the differentiation of heterocysts. The requirement for NtcA indicates a regulatory link between nitrogen nutrition and heterocyst development, so that the latter is integrated into a suite of cellular responses to nitrogen stepdown. In this context, it is of interest that in a derivative of Anabaena sp. strain PCC 7120 that expresses a 2-oxoglutarate permease, 2-oxoglutarate has been reported to increase heterocyst frequency both in nitrate- and no combined nitrogen-containing media [123]. Mutant strains carrying an inactivated ntcA gene show no sign of differentiation upon combined nitrogen deprivation, indicating that the NtcA protein is required at the initiation of the process. Induction of two other positive-acting genes whose products act early in heterocyst development, namely hetR and hetC, has been shown to depend on NtcA. The positive regulatory (and autoregulated) gene hetR encodes a protein that exhibits DNA-binding [75] and autoproteolytic [74] activities in vitro and its mutants do not show any sign of heterocyst differentiation [72]. Induction of hetR, which takes place shortly after combined nitrogen deprivation [72,73], is impaired in ntcA mutant strains of Anabaena sp. strain PCC 7120 [66,124] and N. punctiforme[79], but the basis for such dependence on NtcA is not yet known. Expression of hetR in Anabaena sp. strain PCC 7120 takes place from several promoters [124,125], two of which (those generating tsps located at nucleotides −271 and −728 from the gene) are N-regulated and not used in an ntcA mutant strain but do not show the typical structure of an NtcA-activated promoter or even NtcA-binding sites [124]. Thus, in this case the requirement for NtcA might be indirect.

The Anabaena sp. strain PCC 7120 hetC gene would encode a product similar to proteins of the HlyB family of bacterial ABC exporters, although its putative substrate is unknown [76]. In hetC mutants heterocyst development is arrested at a very early stage, although a delayed pattern of weakly autofluorescent cells in which expression of a hetR::gfp fusion takes place is observable after prolonged nitrogen deprivation [126]. Expression of hetC takes place from a single NtcA-dependent promoter that is activated promptly upon combined nitrogen deprivation [127].

Expression of the ntcA gene itself is induced several-fold during the early steps of heterocyst differentiation in a HetR-dependent and autoregulated manner based on activation of two regulated promoters: one generating tsp −49 that is preferably used in the absence of combined nitrogen and early during heterocyst differentiation, and that is active in mature heterocysts, and another one generating tsp −180 that appears to be transiently used during heterocyst development, but not in mature heterocysts [124,128]. Thus, a mutual dependence is observed in the expression of both regulatory genes ntcA and hetR. Activation of the expression of hetR at the initiation of heterocyst differentiation precedes that of ntcA[124]. This implies that NtcA-mediated activation of hetR expression does not require activation of the expression of the ntcA gene, and makes it conceivable that the HetR-dependent activation of ntcA expression requires previous activation of the expression of hetR. (It should be noted that the requirement of HetR for NtcA function is specific to heterocyst development, since mutation of hetR does not impair growth with nitrate.) Conversely, the NtcA-dependent initiation of hetC transcription is independent of HetR [124]. Because activation of ntcA expression is dependent on HetR, these observations suggest that initiation of hetC transcription does not require HetR-dependent increased expression of the ntcA gene. Additionally, the observation that the NtcA-mediated activation of hetR expression is not impaired in a hetC mutant [126,127] indicates that HetC is not required for activation of hetR expression.

A possible model for a sequence of events of activation of gene expression at the initiation of heterocyst differentiation implies independent activation of the hetR and hetC genes both operated by the initial low levels of NtcA protein already present in the filament exposed to combined nitrogen. Activation of hetR by NtcA would be indirect and enhanced by autoregulation, whereas that of hetC would be direct. Subsequently, the resulting increased cellular levels of the HetR protein would lead to activation of ntcA expression also enhanced by autoregulation (Fig. 2).

Figure 2.

Some events of activation of gene expression at the initiation of heterocyst differentiation. Black arrows represent gene expression from transcription to the corresponding mature protein. Red solid arrows indicate NtcA promoted transcription activation. Dashed arrows indicate a positive action exerted by NtcA (red) or HetR (blue) on gene expression. See the text for further explanations. Different letter sizes in the case of NtcA try to indicate different (not to scale) cellular levels of the protein.

Some genes have also been described that negatively affect heterocyst development. The patS gene encodes a short peptide thought to constitute a diffusible negative signal of differentiation [69]. Overexpression of patS suppresses heterocyst development, whereas mutation of this gene leads to the formation of heterocysts in nitrate-containing medium and Mch in combined nitrogen-free medium [69]. Expression of patS increases during several hours after nitrogen stepdown in a patterned way in the cells that will become heterocysts, and then decreases down to the initial levels [69,70]. Although the way of regulation of patS is currently unknown, the recent report [75] of binding of HetR to its promoter region must be taken into account. PatS seems to be involved in “de novo” heterocyst pattern formation upon combined nitrogen deprivation, by inhibition of the differentiaton of neighbouring cells [69,70].

As is the case for the expression of patS, activation of the expression of both hetR and hetC can be observed to be favoured in those cells of the filament that will develop into heterocysts [73,126] (but see below for the case of activation of the hetC promoter). Whether activation of expression of the ntcA gene also takes place in a patterned way remains to be studied.

Other genes that participate in the early events of heterocyst development and the establishment of the pattern of heterocyst distribution along the cyanobacterial filament have been described. In N. punctiforme, a gene named hetF whose product appears to cooperate with HetR at positive regulation has been identified [79]. Similarly to the situation with hetR, hetF mutants are unable to develop heterocysts, whereas extra copies of hetF induce formation of clusters of heterocysts. Activation of hetR expression in hetF mutants is delayed and not restricted to developing heterocysts, taking place even under nitrogen-replete conditions [79]. The hetF gene is constitutively expressed and its relationship to NtcA, if any, is unknown. Another gene, named hetL, whose overexpression produces Mch even in nitrate-containing medium, has been recently identified in Anabaena sp. strain PCC 7120 [78]. Although a hetL-null mutant shows normal heterocyst development and diazotrophic growth, which might indicate a nonessential role of HetL in the process, hetL overexpression can bypass the suppression of heterocyst differentiation provoked by extra copies of patS, but cannot bypass the requirement for HetC or HetR [78]. Interestingly, hetL overexpression in an ntcA mutant allows signs of initiation of heterocyst development, but differentiation could not proceed and the filaments became highly fragmented [78], consistent with a requirement for NtcA beyond the initial steps of differentiation (see below). Thus, hetL overexpression may have a positive effect on HetR activity or abundance [78], perhaps bypassing the requirement for NtcA of hetR activation. However, it is also possible that HetL is not involved in the regulation of heterocyst differentiation in the wild-type strain [78]. Finally, the hetN gene has been identified whose product would show homology to oxidoreductases involved in fatty acid or polyketide biosynthesis [81]. Overexpression of the hetN gene prevents the patterned activation of hetR under nitrogen deprivation and hence suppresses differentiation [83,129]. However, the observation that in wild-type filaments induction of hetN occurs late in the course of development, and that when expression of this gene is turned off a wild-type initial pattern of heterocysts appears that is later substituted by a Mch phenotype, would imply that HetN plays a role in maintenance of the pattern once it has been established [83].

The effects of manipulation of the expression of the N-control regulator NtcA and of the genes hetR, hetF and patS on heterocyst frequency and spacing pattern can be compared. While strains carrying multiple copies of ntcA (N. punctiforme, [45]) or expressing the ntcA gene from a strong, constitutive promoter (Anabaena sp. strain PCC 7120; E. Olmedo-Verd, A. Herrero, E. Flores and A. M. Muro-Pastor, unpublished) develop heterocysts only in the absence of combined nitrogen, and do so with wild-type spacing pattern and frequency, overexpression of hetR or hetF or mutation of patS leads to the formation of Mch in combined nitrogen-free medium. Moreover, overexpression of hetR or inactivation of patS, but not overexpression of hetF, leads to differentiation of heterocysts in the presence of nitrate [69,72,79,125]. It is tempting to speculate that the action of NtcA could be primarily involved with the triggering and progression of differentiation of a given cell to a functional heterocyst (see below), while the action of the product of hetR (and perhaps also of hetC, hetF and hetL) could be more directly connected to that of the negative factor PatS in determining the spacial distribution of heterocysts and prevention of PatS action inside the differentiating cell. When the cells sense nitrogen deficiency, the balance between the action of positive factors (NtcA, HetR, HetF, HetC, and possibly HetL) and the suppression mediated by PatS (and later by HetN) may lead to the decision of whether or not to differentiate and which particular cell will become a heterocyst.

5Role of NtcA during the progression of heterocyst differentiation and in the mature heterocyst

Induction of a number of genes whose expression is required for the progression of heterocyst development is dependent on an intact hetR gene [130]. The nature of this dependence is currently unknown. Activation of genes whose products are involved in the progression of heterocyst development, and whose relation to NtcA has been studied, has been found to exhibit a requirement for an intact ntcA gene. Because NtcA is required for hetR induction and heterocyst development, impairment of expression of HetR-dependent genes in an ntcA mutant could simply be a consequence of the lack of hetR activation and/or heterocyst differentiation in such a mutant. However, although overexpression of hetR results in heterocyst development even in the presence of nitrate, only in a medium without combined nitrogen are these cells active in nitrogen fixation [125]. This observation implies operation of N regulation beyond induction of hetR, consistent with a direct activation by NtcA of some genes involved in heterocyst function. In fact, recent results from our laboratory have shown that addition of ammonium to a heterocyst-containing culture of Anabaena sp. strain PCC 7120 inhibits accumulation of nifHDK transcripts (E. Olmedo-Verd, A. Herrero, E. Flores and A. M. Muro-Pastor, unpublished). As described below, it has been shown that the NtcA protein is directly involved in transcriptional activation of some genes that are expressed at intermediate stages of heterocyst development or in the mature heterocyst.

Anabaena sp. strain PCC 7120 genes studied to date that act at intermediate stages of heterocyst development and whose expression is activated in a HetR- and NtcA-dependent manner include: the devBCA operon that encodes an ABC transporter involved in the maturation of the heterocyst envelope [59,130,131], the devH gene encoding a putative DNA-binding protein required for N2 fixation in the heterocysts [80], and the cox2 and cox3 operons encoding terminal respiratory oxidases also required for nitrogenase activity in the heterocysts [102]. The devBCA operon is expressed from a N-regulated, NtcA-dependent promoter early upon combined nitrogen deprivation [131]. The prompt activation of this promoter would suggest that it does not require HetR-mediated increased amounts of the NtcA protein. However, the increase of devBCA transcript levels that is detected at intermediate stages of heterocyst development requires HetR in addition to NtcA [131]. The cox2 and cox3 gene clusters are induced at intermediate and late stages, respectively, of heterocyst development and are expressed in mature heterocysts from NtcA- and HetR-dependent promoters [102]. In contrast, direct involvement of NtcA in transcription of devH has not been established yet.

Excision of two intervening DNA elements (the nifD and the fdxN elements, which in Anabaena sp. strain PCC 7120 are of 11 kb and 55 kb, respectively) that in some strains takes place in the course of heterocyst development [132,133] does not take place in hetR (E. Olmedo-Verd, A. Herrero, E. Flores and A. M. Muro-Pastor, unpublished) or ntcA mutants [67]. In Anabaena sp. strain PCC 7120, binding of NtcA to three sites in the region upstream of xisA, which encodes the site-specific recombinase responsible for excision of the nifD element, has been described [134]. The role of these binding sites in regulation of expression of xisA is unknown. It has been hypothesized that binding of NtcA to them could exert a repressor role in vegetative cells [134]. However, excision of the nifD element can be forced, even under nitrogen replete conditions, by increasing the levels of NtcA in a hetR mutant background (E. Olmedo-Verd, A. Herrero, E. Flores and A. M. Muro-Pastor, unpublished), adding to the idea that NtcA might have a positive role on expression of xisA.

Anabaena sp. strain PCC 7120 genes whose induction require NtcA and whose products act in the mature heterocyst include petH (encoding ferredoxin:NADP+ reductase), glnA (encoding glutamine synthetase) and those in the cphBA1 (encoding proteins of cyanophycin metabolism) and nifHDK operons. Ferredoxin:NADP+ reductase, which can contribute to the provision of the reduced ferredoxin required for the nitrogenase reaction, and glutamine synthetase, responsible for the incorporation of the fixed nitrogen into carbon skeletons, are critical for the assimilation of nitrogen in heterocysts. The petH gene is transcribed from two promoters, one constitutive with respect to the nitrogen source and another used in the absence of combined nitrogen and dependent on NtcA. The latter is the main promoter used in heterocysts, but it is also used in a hetR mutant and in the wild type after a nitrogen stepdown before mature heterocysts have developed [104]. The glnA gene is expressed from at least three promoters, one constitutive and two negatively regulated by ammonium and NtcA-dependent [66]. Of these, the one producing RNAI (P1) is the main promoter used in heterocysts and is activated upon combined nitrogen deprivation irrespective of the presence or absence of a functional hetR gene [104].

The nifHDK operon is expressed in Anabaena sp. strain PCC 7120 under oxic conditions exclusively in the heterocysts [135] from a single N-regulated promoter that is not operative in the ntcA[66] or hetR (A. Valladares, A. M. Muro-Pastor, A. Herrero and E. Flores, unpublished) mutants. An additional basis of the requirement for HetR and completion of heterocyst development for expression of the nif genes could originate in a negative effect of oxygen, consistent with the observed requirement of intact cox2 or cox3 genes for expression of nitrogenase activity [102]. In this context, PatB, a DNA-binding protein with a putative ferredoxin-like domain expressed late during development and required for nitrogenase activity expression, may represent a sensor of redox state in the heterocyst [98]. Finally, in heterocysts, promoters PcphB1-1 that directs cotranscription of cphB1 (encoding cyanophycinase) and cphA1 (encoding cyanophycin synthetase) and PcphA1-2 for monocistronic expression of cphA1 are N-regulated and used in an NtcA-dependent manner, although their requirement for HetR has not been investigated [119].

In addition to its role as a transcriptional activator, NtcA appears to act as a repressor of some promoters during the course of heterocyst development (see [5]). Rubisco, encoded in the rbcLXS operon, is not expressed in heterocysts, and in Anabaena sp. strain PCC 7120 NtcA has been shown to bind to two sites in its promoter [136]. At least one of these sites could be a repressor site, since it maps to the region from −12 to +12 with respect to the tsp [136]. The hanA gene encodes the histone-like HU protein that is absent from heterocysts and whose mutation results in a highly pleiotropic phenotype that includes lack of heterocyst development [71,137]. The sequence TGTAN8AACA, that could represent an NtcA binding site, is located 60 nucleotides downstream from a tsp of hanA that has been detected with RNA from ammonium-grown cells [71,137]. Whether this putative NtcA-binding site has a role at suppression of hanA expression remains to be investigated.

The ntcA gene is expressed in fully developed, mature heterocysts [124,128,136,138], and activity of isocitrate dehydrogenase, that produces 2-oxoglutarate, is high in these differentiated cells [64,120]. Since 2-oxoglutarate is a putative positive effector of NtcA, these observations suggest that high levels of active NtcA protein are present in the heterocysts, consistent with an important role of NtcA in gene expression in these differentiated cells.

6A compilation of NtcA-regulated promoters involved in heterocyst development or function

As described above, NtcA is required for activation of expression of genes that are required for the initiation of heterocyst differentiation or for other steps of its development and function. Indeed, with the exception of hetR, every gene required for heterocyst development that has been tested (see below) appears to be directly activated by NtcA.

Activation of expression of some of those genes does not depend on HetR. Because activation of expression of the ntcA gene during heterocyst development is dependent on a functional hetR gene, we suggest that NtcA-dependent transcription of genes that are independent of hetR does not require the increased levels of the NtcA protein that would result from HetR-dependent increased expression of the ntcA gene. Rather, activation of those promoters would be effected at the expense of the initial lower levels of NtcA protein, once metabolically activated upon sensing of combined nitrogen deprivation. This would be the case for the NtcA-dependent promoters of petH and glnA used in the heterocysts but which are activated early after combined nitrogen deprivation not only in the cells that start to differentiate but also in the non-differentiating cells. The same rationale could apply to the promoter of the hetC gene, to the promoter producing tsp −728 of hetR, and possibly to the early use of the promoter of the devBCA operon. (Although it has been described [126] that after prolonged incubation without combined nitrogen a hetC::gfp transcriptional fusion is expressed most strongly in proheterocysts and heterocysts, this would not preclude that the hetC promoter could initially be activated in all cells of the filament. Later, a patterned expression could be established along the filament, e.g. responding to differences in the C/N balance status between different cells, which would determine different levels of active NtcA protein, or to the action of other regulatory proteins.)

On the other hand, activation of some NtcA-dependent genes (or promoters) during intermediate stages of heterocyst development or in the mature heterocyst requires, besides NtcA, a functional hetR gene. These include the ntcA gene itself, the promoter producing tsp −271 of hetR, and the cox2, cox3 and nifHDK operons (see above). For the NtcA-dependent promoters activated late during development, the prediction can be made that a reason for the lack of their early expression could rely upon their requirement for increased amounts of NtcA protein to be activated.

In summary, a hypothetical model for sequential activation of transcription of NtcA-dependent genes during heterocyst development is as presented in Fig. 3. Upon combined nitrogen stepdown, the initial relatively low levels of NtcA protein already present in the combined nitrogen-exposed filament would become activated in response to the change produced in the C to N balance of the cells (e.g., reflected in the increased levels of 2-oxoglutarate). Activated NtcA would then promote transcription from N-regulated, HetR-independent promoters of early activated genes (such as petH, glnA, and possibly also hetC and devBCA), initially in all cells of the filament, and would also promote induction of the hetR gene, known to be localized in cells that will become heterocysts. The resulting increased amounts of the HetR protein would then (acting at an as yet unidentified level) increase the expression of genes that are activated early but are influenced by HetR, such as devBCA and ntcA itself. (At this point, increases in the levels of HetR and NtcA would be potentiated by their autoregulatory character.) The resulting increased levels of the NtcA protein would then be able to promote the use of N-regulated promoters of genes (such as cox2, cox3, nifHDK, and possibly cphBA1 and cphA1) that are activated late during heterocyst development or in the mature heterocysts and require HetR for expression. This would not exclude an additional, non-NtcA-mediated positive effect of HetR on the expression of those late genes.

Figure 3.

Hypothetical model for some steps of sequential activation of gene expression during heterocyst differentiation. Open arrow represents metabolic activation of the NtcA protein. Meaning of other types of arrows are as specified in the legend to Fig. 2. See the text for further explanations. Other putative effects of HetR on transcript accumulation are not depicted. Temporal progression of morphological differentiation (shown on bottom) is only approximate.

From the above considerations, it seems evident that a hierarchy exists in the activation of NtcA-dependent promoters during heterocyst development. To gain insight into the molecular mechanism for this selective NtcA action, a comparison can be made of the structure of the N-regulated, NtcA-dependent promoters of heterocyst genes characterized to date (see Fig. 4).

Figure 4.

Sequence comparisons of NtcA-dependent promoters of genes involved in heterocyst development or function in Anabaena sp. strain PCC 7120. Transcription start sites are indicated by boldface lower case letters (see the text for references). The location of the tsp with respect to the translation start of the corresponding gene is indicated, as is the distance between the −10 box and the NtcA-binding site. The consensus for NtcA-binding site and −10 hexamer of NtcA-dependent promoters [5] are also shown at the bottom of the figure, and those bases of the depicted promoters matching the consensus are indicated in capital boldface. W, A or T; Y, C or T; R, A or G.

In Anabaena sp. strain PCC 7120, the NtcA-dependent promoters of the genes hetC[127], devBCA[131] and glnA (P1) [66] conform to the structure of the canonical NtcA-activated promoter including NtcA-binding sites with the sequence signature GTAN8TAC separated by ca. 22 nucleotides from a −10 box with the consensus sequence TAN3T, a structure similar to that of Class II bacterial activated promoters [15] (Fig. 4, see also Fig. 5). The NtcA-regulated promoters of petH and nifHDK and the NtcA-regulated PcphA1-2 conform to the structure of canonical NtcA-activated promoters but their putative NtcA-binding sites resemble but do not match the sequence signature GTAN8TAC. Instead, the sequence GACN8AAC, in the case of petH, the sequence ACTN8TAC, in the case of nifH[104], and the sequence GTAN8TAG in the case of PcphA1-2 [119] are found 21–22 nucleotides upstream from their respective −10 promoter boxes (Fig. 4, see also Fig. 5). In the three cases, specific in vitro binding of NtcA to DNA fragments including these putative NtcA-binding sequences has been obtained [104,119].

Figure 5.

Schematic representation of different types of NtcA-dependent promoters of genes involved in heterocyst development or function. See text for details. hetR indicate promoters producing tsps −271 and −278; ntcA (P2) represents the promoter producing tsp −180 (see the text for references). Other indicated promoters correspond to those shown in Fig. 4.

Some NtcA-dependent promoters of the ntcA gene and the cphBA1 operon and those of the cox2 and cox3 operons include NtcA-binding boxes, but located upstream from its canonical position in NtcA-activated promoters. The NtcA-activated promoter of the ntcA gene that generates tsp −49 includes the sequence GTAN8AAC, which is strongly similar to the consensus sequence for NtcA binding (and indeed, NtcA footprinting to this sequence has been reported [128]) but is centered at 93.5 nucleotides upstream of that tsp (Fig. 4). On their part, the cox2 and cox3 promoter regions include consensus GTAN8TAC NtcA-binding boxes, but centered at 238.5 and 180.5 bp, respectively, upstream from the tsps (Fig. 4) (A. Valladares, A. Herrero and E. Flores, unpublished). In both cases, in vitro binding of NtcA to DNA fragments containing these sites has been obtained and, in addition, in the case of cox2, mutagenesis of the putative NtcA-binding site changing the GTA triplet to CAT or the TAC triplet to ATG abolishes activation of expression (A. Valladares, A. Herrero and E. Flores, unpublished). Also, the NtcA-dependent promoter PcphB1-1 of the cphBA1 operon includes a consensus GTAN8TAC box, at which NtcA has been shown to bind, centered at −92.5 nucleotides from the corresponding tsp [119] (Fig. 4). These NtcA-activated promoters resemble Class I CAP-dependent promoters in which the binding site for the transcriptional activator is located upstream of the DNA site for RNA polymerase [139], thus representing a new mechanism for NtcA-mediated transcription activation (see Fig. 5).

Finally, no NtcA binding could be demonstrated to, and no recognizable NtcA-binding box could be found in, DNA upstream from any of the two NtcA-dependent tsps of the hetR gene (located at nucleotides −271 and −728 from the gene) [124] or the NtcA-dependent −180 tsp of the ntcA gene, all of which are activated during heterocyst development (see Fig. 5).

The consensual nature of the NtcA-dependent promoters of the glnA, hetC and devBCA genes would be consistent with their early activation during heterocyst differentiation. NtcA would be expected to exhibit high affinity for these promoters, not requiring for binding the HetR-mediated increase of the cellular levels of the NtcA protein. On the other hand, the imperfect NtcA-binding box of the nifH gene (for which NtcA indeed shows a relatively low affinity in vitro) would be consistent with a late activation based on the requirement for increased amounts of NtcA protein (and perhaps also of other late-acting factors). With regard to PcphA1-2, although NtcA exhibits in vitro a high affinity for binding to it, interference by an overlapping promoter could explain its in vivo late activation [119]. It would remain to be interpreted how the NtcA-dependent promoter of the petH gene, which exhibits a non-consensus NtcA-binding box (with poor in vitro NtcA binding [104]), is activated early. On the other hand, we currently cannot make a prediction on how the order of activation of Class I- versus Class II-type NtcA-activated promoters is established. Finally, NtcA-mediated regulation of the expression of hetR and ntcA promoters that do not show hints of direct NtcA binding could involve another NtcA-dependent factor yet to be identified.

7Concluding remarks and prospects

Some filamentous cyanobacteria can undergo a suite of cellular differentiation processes that permit their better adaptation to changing environmental conditions. Whereas the differentiation of both hormogonia and akinetes can be triggered by a variety of environmental cues, the principal factor leading to the differentiation of heterocysts, which allow the cyanobacterium to make use of atmospheric nitrogen as a source of nitrogen, is lack of combined nitrogen. The molecular basis for the differentiation process has been more thoroughly studied in the case of heterocyst development. The decision of whether to differentiate a heterocyst seems to be reached through the interplay between a number of positively and negatively acting factors, some of which have been identified in recent years. The transcription factor NtcA, that operates global N control in cyanobacteria by regulating the expression of multiple genes involved in nitrogen assimilation, has a crucial role in the triggering of heterocyst differentiation by perceiving the nitrogen status of the cell and initiating a series of gene promoter activation events that sustain the differentiation process. This cascade of gene activation events includes activation of the ntcA gene itself and of another positive-acting gene, hetR, whose product is also pivotal for the initiation of differentiation. HetR could be a regulator not exclusively responding to the nitrogen status, but able to integrate information of more than one environmental factor and/or cellular condition. This would be evident if hetR were required for akinete development, a process that could be triggered by environmental cues not related to nitrogen nutrition (see above). In this scenario, NtcA would be the factor transmitting to HetR information about the nitrogen status of the cell to engage it in initiation of heterocyst development. Gene activation events at the initiation of heterocyst differentiation can also be influenced by the action of some other positive (such as the hetC gene product) or negative (such as PatS) elements. While the action of NtcA as a transcriptional activator (or repressor) has been reasonably well established, the mechanism of action of some other factors decisive for heterocyst development, including HetR, is currently unknown and should be the subject of research in the near future. In this context, the recent report of DNA-binding activity of HetR can orient future research.

The action of NtcA is not only required at the initiation of heterocyst differentiation, but also for the continuation of the process and for the function of the mature heterocyst. This requirement is based on the role of NtcA as a transcriptional activator of structural, and possibly also regulatory, genes whose products participate throughout the differentiation process or in the distinctive metabolism of the heterocyst. These genes may or may not be specific for these cells, since some NtcA-activated genes that encode products active in the heterocyst are also activated by NtcA in vegetative cells under conditions of combined nitrogen deprivation. The molecular basis for the selective action of NtcA in the activation of certain, and not other, NtcA-dependent genes at precise points during the process of heterocyst development is unknown, but the cooperation between NtcA and other regulatory factors that may accumulate at specific stages of the developmental process could have a role. Also, the differences found in the features of the NtcA-activated promoters are expected to have a role in the establishment of a hierarchy of activation of NtcA-dependent genes, possibly by influencing the level of active NtcA required for their activation. Thus, the study of how changes in the sequence of the NtcA-binding box and how its position in the NtcA-activated promoters influence the affinity of NtcA, of how NtcA binding could be affected by the action of other possible regulatory factors, and of how the cellular levels of active NtcA protein change during the course of heterocyst development, together with the effects of 2-oxoglutarate and the PII protein, can be regarded as crucial areas of research in this field.

Acknowledgements

We thank Dr. José Enrique Frías for help with photographs shown in Fig. 1 and Silvia Picossi for help with the preparation of the manuscript. Work in the authors' laboratory is currently supported by Grants No. BMC2001-0509 and BMC2002-03902 from the Ministerio de Ciencia y Tecnología (Spain) and by Plan Andaluz de Investigación, research group CVI129. Strain 9v shown in Fig. 1 was isolated in the context of Project ICA4-CT-2001-10058 from The European Community.

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