The NtcA Transcriptional Regulator

ntcA was first identified in the unicellular cyanobacterium Synechococcus sp. PCC 7942 as a gene whose mutation caused the inability to derepress N assimilation proteins in response to ammonium withdrawal; the Synechococcus ntcA mutant can grow with ammonium but not with nitrate as the N source (Vega-Palas et al. 1990). Northern analysis has confirmed that a number of genes or operons in Synechococcus sp. PCC 7942 (e.g. amtl, the nir operon) are not expressed in an ntcA mutant, and primer extension analysis permitted to identify transcription start points whose use requires an intact NtcA protein (reviewed in Herrero et al. 2001). This observation, together with the regulatory behavior of revertants of the original ntcA mutant (Vega-Palas et al. 1990), allowed identification of NtcA as a positive-acting transcription factor for those genes or operons. The ntcA gene is widespread in cyanobacteria (Frias et al. 1993), and ntcA mutants have also been reported for the heterocyst-forming cyanobacteria Anabaena sp. PCC 7120 (Frias et al. 1994; Wei et al. 1994), Anabaena variabilis (Thiel, Pratte 2001), and Nostoc punctiforme (Wong, Meeks 2001). None of these mutants can develop heterocysts, which suggests that NtcA also links heterocyst development to the environmental cue of N limitation.

The deduced amino acid sequence of the NtcA protein shows a high similarity in all the cyanobacteria for which the ntcA gene has been identified and sequenced. NtcA consists of -220 amino acid residues and is homologous to proteins of the CAP family of bacterial transcriptional regulators (Herrero et al. 2001). NtcA bears three strongly conserved regions: a 73-amino acid N-terminal region which corresponds to the CAP region of (5-roll structure, a 39-amino acid central region that may correspond to the region of CAP involved in protein dimerization, and a 24-amino acid C-terminal region exhibiting features of a helix-turn-helix structure characteristic of DNA binding domains (Herrero et al. 2001). Band-shift assays have enabled to identify DNA fragments carrying NtcA-binding sites, and DNase I footprinting analysis has identified more precisely the binding site for NtcA upstream from a number of NtcA-regulated genes (Luque et al. 1994; Frias et al. 2000). Scrutiny of the NtcA binding sites in the promoters of twenty-one NtcA-activated genes (reviewed in Herrero et al. 2001) has shown that their sequences correspond to the following consensus sequence.

The strongly conserved GTANgTAC sequence is located in these promoters ~22 nucleotides upstream from a -10 box with the sequence TAN3T, and the spacing between the -10 box and the transcription start point is ~6 nucleotides. Thus, the characterized NtcA-dependent promoters are similar to the class II CAP-activated promoters, where the binding site for the regulator is centered at about —41.5 with respect to the transcription start point.

The sequence of an NtcA-binding site determines the affinity of NtcA for such binding site. Thus, different dissociation constants (¡(¿) have been found for the binding of NtcA to different NtcA-binding sites, ranging from about 33 nM to about 1.4 |iM for the NtcA-binding sites of the Synechococcus glnA and glnB gene promoters, respectively (M.F. Vazquez-Bermudez, unpublished). It is possible that the affinity of NtcA towards its binding sites in different regulated promoters helps to establish a hierarchy of gene expression under N limitation. Additionally, we have used the determination of the Ka of NtcA towards some mutated NtcA-binding sites to test the importance of certain nucleotides in the NtcA-binding site. The data obtained have corroborated the essential role of the most conserved nucleotides in establishing the binding affinity of NtcA.

5. How is NtcA Regulated?

In Synechococcus sp. PCC 7942, ntcA is a positively autoregulated gene. While in the presence of ammonium it is expressed at a low level from a constitutive promoter, after ammonium withdrawal it is induced being transcribed from a canonical NtcA-dependent promoter (Luque et al. 1994). It appears that the low level of NtcA protein present in ammonium-grown cells is able to activate transcription upon removal of ammonium and, therefore, that the NtcA protein is subjected to N-regulation. Consistent with this hypothesis, overexpression of the ntcA gene in Synechococcus sp. PCC 7942 and Anabaena sp. PCC 7120 does not result in the constitutive expression of genes activated by NtcA (I. Luque, M.F. Vazquez-Bermudez, J.E. Frias, unpublished).

We have observed that 2-oxoglutarate stimulates the binding in vitro of NtcA to the Synechococcus glnA promoter, but not to other Synechococcus promoters like that of the nir operon (M.F. Vazquez-Bermudez, unpublished). The Kd for NtcA binding to the glnA promoter in the presence of 2-oxoglutarate is about four-fold lower than in its absence. Pn, the glnB gene product, is a C/N balance signal transduction protein that is widespread in bacteria and has also been characterized in cyanobacteria (Arcondeguy et al. 2001). Although previous studies indicated that N-regulation of expression of the Synechococcus nir operon proceeds normally in a glnB mutant (Lee et al. 1998), we have now observed that induction of amtl, another NtcA-dependent gene, is impaired in a Synechococcus glnB mutant (J. Paz-Yepes, unpublished). We are currently investigating the possible role of 2-oxoglutarate and the Pn protein as additional elements for transcriptional N-control in cyanobacteria. The ntcA gene has been expressed in E. coli and, using constructs designed to report repression or activation of gene expression, we have observed repression provoked by NtcA as well as NtcA-dependent gene expression in this heterologous system (P. Barraillé, unpublished). These results open the possibility of studying aspects of the NtcA function in a well-characterized system like E. coli.

6. Role of NtcA in Heterocyst Development and Function

Heterocyst development takes place in response to N deprivation and, as mentioned above, requires NtcA. After NtcA, a number of genes whose products appear to act as early regulators of the process of development, such as hetR, hetC and hetP, are induced (Wolk 2000). Numerous genes that are required for the morphological and biochemical differentiation of the heterocyst are then expressed. These include genes encoding proteins involved in the biosynthesis of the heterocyst envelope polysaccharide (hep genes) and glycolipid (devBCA, hgl genes) layers, as well as genes encoding enzymes like nitrogenase, glutamine synthetase (glnA) or ferredoxin-NADP+ reductase (petH) that function in the mature heterocyst (Wolk 2000).

The expressions of the hetC gene and of the devBCA operon are dependent on NtcA and take place from NtcA-type canonical promoters, and the binding of NtcA to DNA fragments containing these promoters has been shown in vitro (Muro-Pastor et al. 1999; Fiedler et al. 2001). In the mature heterocyst, the glnA gene is transcribed mainly from the so-called Pi promoter (A. Valladares, A. M. Muro-Pastor, unpublished), a well-characterized NtcA-dependent promoter (Frías et al. 1994). These results indicate that in addition to being required for the process of heterocyst development to start, NtcA also has an active role in gene expression during the differentiation of the heterocyst and in the mature heterocyst. It appears therefore that N-control may be operative at different steps of heterocyst development and even in the mature heterocyst.

7. Novel, Non-Canonical NtcA-Dependent Promoters

The NtcA-dependent promoters discussed above all conform to the canonical "class II" structure on which NtcA likely interacts directly with the RNA polymerase. However, a number of cyanobacterial genes have recently been characterized whose expression is NtcA-dependent but for which a canonical NtcA-type promoter is not evident. Among these genes, there are some that may be regulated only indirectly by NtcA (e.g. the hetR gene), but there are others whose promoters contain some kind of NtcA-binding site. Thus, the promoters of the cox2 (encoding a cytochrome c oxidase) and cph (encoding enzymes of cyanophycin metabolism) operons of Anabaena sp. PCC 7120 contain standard NtcA-binding sites which are situated farther upstream from the -41.5 position (A. Valladares, S. Picossi, unpublished). On the other hand, the promoters of the petH gene and of the nifHDK operon show, at about position -41.5, sequences that resemble but do not match that of the standard NtcA-binding site (Valladares et al. 1999). In these promoters, the participation of other regulators or transcription factors in NtcA-dependent activation of gene expression is an interesting possibility to be addressed in the future.

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