Mutual dependence of the expression of the cell differentiation regulatory protein HetR and the global nitrogen regulator NtcA during heterocyst development

1 Mutual dependence of the expression of the cell differentiation regulatory protein HetR and the global nitrogen regulator NtcA during heterocyst development Alicia M. Muro-Pastor*, Ana Valladares, Enrique Flores, and Antonia Herrero Instituto de Bioquímica Vegetal y Fotosíntesis, Centro de Investigaciones Científicas Isla de la Cartuja, CSIC-Universidad de Sevilla, Avda. Américo Vespucio s/n, E41092 Sevilla, Spain *For correspondence. Phone: 34-95-4489523. Fax: 34-95-4460065. E-mail: [email protected]. Key words: hetR, ntcA, heterocyst differentiation, cyanobacteria, Anabaena sp. Running title: hetR and ntcA expression in Anabaena 2 Summary Heterocyst differentiation in the cyanobacterium Anabaena sp. strain PCC 7120 depends on both the global nitrogen regulator NtcA and the cell differentiation regulatory protein HetR, and induction of hetR upon nitrogen step-down depends on NtcA. The use of two of the four transcription start points (tsps) described for the hetR gene (those located at positions -728 and -271) was found to be dependent on NtcA, and the use of the tsp located at position -271 was also dependent on HetR. Thus, autoregulation of hetR could take place via the activation of transcription from this tsp. Expression of ntcA in nitrogen-fixing cultures was higher than in cells growing in the presence of ammonium or nitrate, and high expression of ntcA under nitrogen deficiency resulted from an increased use of tsps located at positions -180 and -49. The induction of the use of these tsps did not take place in ntcA or hetR mutant strains. These results indicate a mutual dependency in the induction of the regulatory genes hetR and ntcA that takes place in response to nitrogen step-down in Anabaena cells. Expression of the hetC gene, which is also involved in the early steps of heterocyst differentiation, from its NtcA-dependent tsp was however not dependent on HetR. 3 Introduction Cyanobacteria are a widely distributed group of phototrophic bacteria that carry out oxygenic photosynthesis. Cyanobacteria are able to use different nitrogen sources, including nitrate and ammonium, and many can also fix atmospheric nitrogen. Ammonium is used in preference over nitrate, which is used in preference over dinitrogen (Flores and Herrero, 1994; Herrero et al., 2001). At the molecular level, this possibility of choice is based on a nutritional repression exerted by ammonium on the expression of genes involved in the assimilation of alternative nitrogen sources, including atmospheric nitrogen, and ntcA has been identified as a gene that encodes a transcriptional regulator operating global nitrogen control that is widespread in cyanobacteria (Frías et al., 1993; Luque et al., 1994; Herrero et al., 2001). The NtcA protein belongs to the CRP family of bacterial regulators and bears close to its Cterminal end a helix-turn-helix motif for interaction with DNA (Vega-Palas et al., 1992). NtcA binds to specific sites in the promoter region of regulated genes involved in nitrogen assimilation and activates their expression in response to ammonium withdrawal (Luque et al., 1994). The structure of the consensus cyanobacterial NtcAactivated promoter comprises a -10 box in the form TAN3T and an NtcA-binding site containing the sequence signature GTAN8TAC that is located 20-23 nucleotides upstream from the -10 box and appears to substitute for the -35 box that would be present in promoters similar to the canonical E. coli σ70 promoters (Luque et al., 1994; Herrero et al., 2001). The ability to fix atmospheric nitrogen depends on the oxygen-sensitive enzyme nitrogenase. Some filamentous cyanobacteria are able to differentiate, under conditions of aerobiosis and combined nitrogen deprivation, cells specialized in nitrogen fixation called heterocysts that develop from vegetative cells located at semiregular intervals in the filament (Buikema and Haselkorn, 1993; Wolk, 2000). 4 Heterocysts provide the microaerobic environment required for nitrogenase activity. The differentiation process begins shortly after nitrogen deprivation and, under our experimental conditions, requires about 24 h to complete. Heterocysts differ from vegetative cells in many structural and functional features that turn them into efficient factories for nitrogen fixation. At the molecular level, the differential physiology of the heterocyst is supported by a pattern of gene expression that largely differs from that taking place in vegetative cells (Buikema and Haselkorn, 1993; Wolk, 2000). Heterocyst development requires the product of the regulatory gene hetR (Buikema and Haselkorn, 1991; Black et al., 1993), which is not required for growth using combined nitrogen and whose expression is induced shortly after nitrogen deprivation. Induction of the expression of hetR does not take place in a ntcA mutant (Frías et al., 1994). The NtcA protein is required for heterocyst development (Frías et al., 1994; Wei et al., 1994) as well as for the function of mature heterocysts (Herrero et al., 2001) and appears to link nitrogen deficiency and heterocyst differentiation in the context of a hierarchy of the use of different nitrogen sources. Another earlyacting heterocyst development gene is hetC, whose product shows homology to ATP-binding cassette (ABC) transporters (Khudyakov and Wolk, 1997). Primer extension analysis has been carried out to define the transcription start points (tsps) for the ntcA, hetR, and hetC genes in Anabaena sp. strain PCC 7120. Three extension products that localize RNA 5’ ends at positions -49, -180, and -190 with respect to the ntcA translation start have been described, and DNA fragments carrying the regions around the -49 and -180/-190 sites have been found to exhibit promoter activity tested with a lacZ reporter, indicating the presence of at least two different promoters in the ntcA upstream region (Ramasubramaniam et al., 1996). Additionally, at least the -49 site is preceded by a putative -10 promoter hexamer. Four extension products have been reported for hetR that localize RNA 5’ ends at 5 -184, -271, -696, and -728 nucleotides with respect to the translation start of this gene (Buikema and Haselkorn, 2001). Although no independent evidence for promoter activity upstream from these sites has been presented, the fact that they may correspond to hetR transcripts of 1.4/1.5 and 1.9 kb detected by Northern analysis (Buikema and Haselkorn, 1991) suggests that those 5’ ends do not originate from primer extension artifacts. Additionally, because the long (1.9 kb) and short (1.4/1.5 kb) hetR transcripts show different expression patterns, they likely originate from different promoters. Scrutiny of the nucleotide sequences upstream from those RNA 5’ ends shows that the four of them are preceded, with a spacing of 6 or 8 nt, by putative -10 promoter hexamers, consistent with the possibility that they represent true tsps. Finally, the hetC gene has been found to be expressed from an NtcAactivated promoter that generates a tsp at -571 with respect to the translation start of the gene (Muro-Pastor et al., 1999). In an attempt to determine the relationships between the products of hetR and ntcA during the triggering of heterocyst differentiation, we have studied the accumulation of their transcripts in strains with different genetic backgrounds (i.e., wild type, ntcA, hetR). We have also analyzed the use of transcription start sites for both genes in the ntcA and hetR strains as well as the effect of hetR inactivation on the transcription of hetC. Results Levels of ntcA expression The NtcA protein is dispensable for growth using ammonium as a nitrogen source. However, it is necessary for growth using nitrate or atmospheric nitrogen. Fig. 1 shows that cultures of Anabaena sp. strain PCC 7120 growing under different nitrogen regimes exhibited different levels of the ntcA mRNA. As previously 6 described (Wei et al., 1994; Ramasubramanian et al., 1996), ntcA was expressed as several transcripts in the range 0.8-1.4 kb. The ntcA expression level was similar in cells growing in the presence of ammonium or nitrate. However, when the cells were grown under diazotrophic conditions, ntcA was expressed at a higher level (2.6-fold the level of transcripts found in ammonium-containing medium for the experiment shown in Fig. 1). This observation might suggest that the level of ntcA transcription required for growth under diazotrophic conditions is higher than that necessary for growth on nitrate. Although in both cases there is a strict requirement for NtcA, diazotrophic growth of Anabaena filaments involves the development of nitrogenfixing heterocysts. Time-course of induction of ntcA and hetR in response to nitrogen deprivation In Anabaena sp. strain PCC 7120, expression of hetR is induced shortly after nitrogen deprivation (Buikema and Haselkorn, 1991; Black et al., 1993) in an NtcAdependent manner (Frías et al., 1994). Similar results have been described for the symbiotically competent Nostoc punctiforme strain ATCC 29133 (Wong and Meeks, 2001). The precise time course of the induction of expression of ntcA and hetR depends on the experimental conditions, so that results from different laboratories may not be strictly comparable. We have carried out Northern experiments in order to compare the time course of induction of the two genes, ntcA and hetR, when the cells are subjected to nitrogen deprivation. Under our experimental conditions, induction of expression of hetR took place, in wild type cells, after less than three hours of nitrogen deficiency, and expression was highest between 4.5 and, at least, 9 h (Fig. 2A, upper panels, left side in both cases). As expected (Frías et al., 1994), there was no increase in the abundance of the hetR transcripts in response to nitrogen deprivation in an ntcA background (strain CSE2). Thus, in the ntcA mutant, there was 7 no increase in the abundance of the 1.4/1.5-kb hetR transcripts already observed in the presence of ammonium and essentially no 1.9-kb transcripts appeared (Fig. 2A, upper right panel, right side, and Fig. 2C). Induction of ntcA was observed to start sometime between 4.5 and 6 h of nitrogen deficiency (Fig. 2A, central panels, left side in both cases), a time frame in which hetR expression was already induced. Thus, the initial induction of expression of hetR in response to nitrogen deprivation precedes in time that of ntcA. This observation suggests that the level of ntcA transcription that takes place in the presence of ammonium, a condition in which NtcA is not required for growth, is sufficient to induce hetR in response to nitrogen deficiency. The results shown in Fig. 2A (left central panel, right side) and Fig. 2B additionally indicate that the induction of expression of ntcA in response to nitrogen deficiency depends on HetR, since it does not take place in a hetR mutant background (strain 216). This observation, together with the results shown in Fig. 1, indicates that the increased expression of ntcA observed in diazotrophic cultures is not required for the initial stages of heterocyst development but rather for later stages once the induction of expression of the cell differentiation regulatory gene hetR has taken place. hetR promoters Both the hetR (Buikema and Haselkorn 2001) and ntcA (Ramasubramanian et al., 1996) genes are transcribed from complex promoters with multiple transcription start sites. To determine the regulation of expression from each of those tsps, primer extension experiments were carried out with RNA isolated from wild type Anabaena sp. strain PCC 7120, the ntcA mutant CSE2 (Frías et al., 1994), and two hetR strains, insertional mutant DR884a (Black et al., 1993) and strain 216, which bears a hetR 8 point mutation, S179A (Buikema and Haselkorn 1991). The four putative tsps described for the hetR gene in Anabaena sp. strain PCC 7120 have been reported to be nitrogen regulated (Buikema and Haselkorn, 2001). To analyze the hetR tsps, we isolated RNA from wild type and mutant cells grown in the presence of ammonium or grown with ammonium and subjected to nitrogen starvation for 6 h. Results shown in Fig. 3C, D indicate that, under our experimental conditions, the use of the initiation site located at position -184 was not nitrogen-regulated, since this site was used in the wild type both in cells grown in ammonium-containing media and in cells subjected to nitrogen deprivation for 6 h, as well as in the ntcA and hetR mutant strains. However, in the wild type, the use of the other three initiation sites (located at -728, -696, and -271) was activated upon nitrogen step-down (Fig. 3). The tsp located at position -271 was not used in the ntcA strain CSE2 (Fig. 3C), indicating that its use is dependent on the nitrogen regulator NtcA. This inducible tsp is also dependent on HetR, since it was very weakly used in any of the two hetR strains, the insertional mutant (strain DR884a) and the point mutant (strain 216) (Fig. 3D). Thus, in this regard, both hetR mutations have the same effect indicating that the use of the tsp located at position -271 requires a functional HetR protein, consistent with the autoregulatory character of hetR (Black et al., 1993; Buikema and Haselkorn, 2001). Induction of the use of the tsp located at position -696 was independent of both NtcA and HetR, whereas the induction of the tsp located at position -728 was severely reduced in the ntcA mutant but unaffected by mutations in hetR (Fig. 3A, B). The sequences of the region upstream from the two NtcAregulated tsps (-728 and -271) do not match the sequences found in canonical NtcAactivated promoters, and band-shift assays containing purified NtcA protein and fragments corresponding to the promoter region of hetR have failed to show any binding of NtcA to those promoters in vitro (not shown). Thus, the NtcA dependence 9 of the utilization of both tsps may be indirect, that is, mediated by other protein(s) that could, in turn, be regulated by NtcA. The hetR transcript found in the presence of ammonium (Fig. 2A, upper right panel) originates, most likely, from the tsp located at position -184, which is constitutive. Expression from the -271 tsp, which is dependent on HetR and originates the 1.5 kb transcript, would account for the autoregulatory character of hetR (Black et al., 1993; Buikema and Haselkorn, 2001). Finally, the large, 1.9-kb transcript, which is almost not induced in the ntcA mutant, may have its origin mostly at the NtcA-dependent -728 tsp. ntcA promoters In order to study, at the level of transcription initiation, the effects of hetR and ntcA mutations on ntcA expression, primer extension experiments with RNA isolated from cells subjected to different nitrogen regimes were carried out (Fig. 4). Under our experimental conditions, extension products ending at positions -49, -136 and -180 were found. We only detected a very faint band that could correspond to the tsp previously found at position -190 (Ramasubramanian et al., 1996). The extension product whose 5’ end located at position -136 appeared to be constitutive, being used in nitrateor ammonium-containing media as well as in cells subjected to nitrogen deficiency or in diazotrophic cultures, and it was also observed in the hetR and the ntcA mutant backgrounds (Fig. 4A). Centered 10.5 nucleotides upstream of the -136 site, a putative -10 promoter box (TATCAT) is present, supporting the assignation of the -136 site as an additional transcription start for the ntcA gene. The tsp located at position -49 was also observed under all conditions, but its use was induced under nitrogen deprivation (Fig. 4A; see also Ramasubramanian et al., 1996). The putative tsp located at position -180, on the contrary, was only used in the absence of combined nitrogen and exhibited a strong, transient induction 16 start of hetR; Buikema and Haselkorn, 2001). In the case of the ntcA gene, the oligonucleotides used were NA1 (complementary to positions +59 to +40 relative to the translation start of ntcA) and NA7 (complementary to positions +14 to -4 relative to the translation start of ntcA). In the case of the hetC gene, the oligonucleotide used was HC2 (complementary to positions -411 to -431 relative to the translation start of hetC). Northern blot analysis was carried out as described (Muro-Pastor et al., 1999). The hetR probe was a 703-bp Hae II fragment containing most of the hetR gene (Buikema and Haselkorn, 1991). The ntcA probe was a 690-bp Nco I-Sal I fragment from pCSAM61 (a plasmid containing the whole Anabaena ntcA gene cloned between the Nco I and BamH I sites of expression vector pTrc99A [Amersham Pharmacia]). Images of radioactive filters and gels were obtained and quantified with a Cyclone storage phosphor system and OptiQuant image analysis software (Packard). 17 Acknowledgements We thank C. P. Wolk for strain DR884a, W. J. Buikema and R. Haselkorn for sharing unpublished results, strain 216 and plasmid pWB216S2.4, J. E. Frías for plasmids and useful discussion, and S. Picossi for some RNA samples. This work was supported by grants from the Dirección General de Enseñanza Superior e Investigación Científica (PB97-1137) and Dirección General de Investigación (BMC 2001-0509). 18 References Ausubel, F.M., Brent, R., Kingston, R.E., Moore, D.D., Seidman, J.G., Smith, J.A., and Struhl, K. (eds) (2001) Current Protocols in Molecular Biology, Greene Publishing and Wiley-Interscience, New York, USA. Black, T.A., Cai, Y., and 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. 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Shimkets (ed.), Prokaryotic Development. American Society for Microbiology, Washington, DC. Wong, F.C.Y., and Meeks, J.C. (2001) The hetF gene product is essential to heterocyst differentiation and affects HetR function in the cyanobacterium Nostoc punctiforme. J Bacteriol 183: 2654-2661. 21 Figure legends Fig. 1. Northern blot analysis of expression of the ntcA gene in Anabaena sp. strain PCC 7120. RNA was isolated from cells grown in the presence of ammonium (lane NH4+) or nitrate (lane NO3-) or from diazotrophic cultures (lane N2). Samples contained 70 µg of RNA. Hybridizations to a ntcA probe (upper panel) or to a rnpB (Vioque, 1997) probe as loading and transfer control (lower panel) were performed. Quantification of the ntcA transcripts was normalized using the rnpB signal, and the transcript level was divided by the value for the NH4+- grown culture (arbitrarily set to 1.0). Values obtained were 2.6 (N2) and 1.2 (NO3-). Arrowheads point to the different transcripts described for the ntcA gene. Fig. 2. Northern blot analysis of expression of the ntcA and hetR genes in Anabaena sp. strain PCC 7120, the ntcA strain CSE2 and the hetR strain 216. RNA was isolated from ammonium-grown cells or from ammonium-grown cells subjected to nitrogen deficiency for the number of hours indicated in each case. Samples in the filters shown in the left and right panels correspond to different experiments including the wild type strain in both cases. Samples contained 50 µg of RNA. (A) The filters were hybridized to a hetR probe (upper panels), an ntcA probe (central panels) or an rnpB probe (Vioque, 1997) (lower panels). Note that the absence of signal in the right side of central right panel (strain CSE2, ntcA probe) can be due to disruption of the ntcA gene in strain CSE2 that might render the ntcA transcript unstable or not detectable with the ntcA probe used. (B) Relative abundance of the ntcA transcript in the wild type and the hetR mutant. Signals (entire lane, including the smear) in the central left panel of Fig. 2A were quantified and normalized using the rnpB signal (lower left panel). The highest value was arbitrarily set to 100. (C) Relative abundance of the hetR transcript in the wild type and the ntcA mutant. Signals (entire 22 lane, including the smear) in the upper right panel of Fig. 2A were quantified and normalized using the rnpB signal (lower right panel). The highest value was arbitrarily set to 100. WT, wild type strain PCC 7120. Arrowheads point to the different transcripts described for the hetR and ntcA genes. Fig. 3. Primer extension analysis of the expression of hetR in Anabaena sp. strain PCC 7120, the ntcA strain CSE2 and hetR strains DR884a and 216. RNA was isolated from cells grown with ammonium (lanes labeled +) or grown with ammonium and deprived of combined nitrogen for 6 h (lanes labeled -). Oligonucleotides used as primers were HR2 (A, B), HR3 (C), and HR7 (D). Similar results were obtained for the -696 and -728 tsps (shown in panels A, B) using oligonucleotide hetRPEX3 (not shown). Sequencing ladders were generated with the corresponding oligonucleotides and plasmid pWB216S2.4 (Buikema and Haselkorn, 1991). The putative tsps are indicated by arrowheads. WT, wild type strain PCC 7120. Fig. 4. Primer extension analysis of the expression of ntcA in Anabaena sp. PCC 7120, the ntcA strain CSE2 and the hetR strain DR884a. (A) RNA was isolated from cells grown with ammonium (lane labeled 0), or grown with ammonium and deprived of combined nitrogen for 6, 12 or 24 hours. (B) RNA was isolated from cells grown with ammonium (lane labeled 0), from cells grown with ammonium and deprived of combined nitrogen for 9 h or from isolated heterocysts (lane labeled H). (C) RNA was isolated from cells grown in the presence of ammonium (NH4+), atmospheric nitrogen (N2) or nitrate (NO3-). Oligonucleotides used as primers were NA1 (A, B) and NA7 (C). Sequencing ladders were generated with the corresponding oligonucleotides and ntcA-containing plasmid pCSE36 (J. E. Frías, unpublished). The putative tsps are indicated by arrowheads. WT, wild type strain PCC 7120. Note: as a routine control 23 of the lack of contamination of RNA from heterocysts with RNA from vegetative cells, we carry out hybridizations to rbcL, encoding the large subunit of ribulose-1,5bisphosphate carboxylase/oxygenase that is expressed only in vegetative cells (Elhai and Wolk, 1990). Hybridization to nifH, encoding nitrogenase reductase that is expressed only in the mature heterocyst, is used as a control of the quality of the heterocysts RNA. The RNA preparation from isolated heterocysts used in the experiment shown in panel B did not show hybridization to a probe of the rbcL gene, but did show hybridization to a probe of the heterocyst-specific nifH gene (not shown). Fig. 5. Time course of the expression of the hetC gene in Anabaena sp. strain PCC 7120 and the hetR strain DR884a upon nitrogen step-down. Primer extension assays were carried out with RNA isolated from cells grown with ammonium (lanes labeled 0) or grown with ammonium and incubated in combined nitrogen-free medium for 3, 6, 9, 12 or 24 h. The oligonucleotide used as primer was HC2. The sequencing ladder was generated with the corresponding oligonucleotide and plasmid pCSAM83 (Muro-Pastor et al., 1999). The tsp located at position -571 is indicated by an arrowhead. WT, wild type strain PCC 7120. N2NO3-NH4+ Muro-Pastor et al., FIG 1 TOP 1.4 kb 0.8 kb 1.1 kb 0 1 4.5 7.5 9 24 0 1 4.5 7.5 9 24 WT CSE2 0 3 6 9 24 0 3 6 9 24 WT 216 1.5/1.4 kb 1.9 kb hetR ntcA rnpB Muro-Pastor et al., FIG 2 TOP A WT CSE2 0 20 40 60 80 100 0 3 6 9 24 0 3 6 9 24 ntcA 0 1 4.5 7.5 9 24 0 1 4.5 7.5 9 24 hetR WT 216 C B mRNA (arbitrary units) mRNA (arbitrary units) 0.8 kb 1.4 kb 1.0 kb 0 20 40 60 80 100 time (h) time (h)