A combinatorial strategy of alternative promoter use during differentiation of a heterocystous cyanobacterium

1 A combinatorial strategy of alternative promoter use during differentiation of a heterocystous cyanobacteria Alicia M. Muro-Pastor*, Manuel Brenes-Álvarez and Agustín Vioque Instituto de Bioquímica Vegetal y Fotosíntesis, Consejo Superior de Investigaciones Científicas and Universidad de Sevilla, E-41092 Sevilla, Spain *Correspondence to: Alicia M. Muro-Pastor Instituto de Bioquímica Vegetal y Fotosíntesis Centro de Investigaciones Científicas Isla de la Cartuja Américo Vespucio 49, E-41092, Sevilla, España (+34) 954489521 [email protected] Running title: Alternative promoters in heterocyst differentiation 2 Originality-significance statement In response to nitrogen deficiency, heterocyst differentiation involves the selection of certain individual cells along cyanobacterial filaments to undergo a complete transformation into a different, nitrogen-fixing cell-type. Such transformation relies on precise regulation of transcripts that might be required, although at different levels, both in vegetative cells and in cells undergoing differentiation as heterocysts. We have noticed that some housekeeping genes are transcribed from combinations of transcriptional starts that respond to nitrogen deficiency with ostensibly opposite behavior. Here we propose that such combinations of promoters are in fact the result of combinatorial promoter use in the two cell types of cyanobacterial filaments. Using the example of the gene encoding the housekeeping sigma factor SigA, we show that at the same time its major promoter is repressed as a consequence of nutritional deprivation, a second, heterocyst-specific promoter is induced to bypass repression specifically in those cells that need to continue actively transcribing. This combinatorial strategy provides additional regulatory possibilities to achieve precise cell-specific levels of transcripts. 3 Summary Heterocystous cyanobacteria such as Nostoc sp. are filamentous photosynthetic organisms that, in response to nitrogen deficiency, undergo a differentiation process transforming certain, semi-regularly spaced cells into heterocysts, devoted to nitrogen fixation. During transition to a nitrogen-fixing regime, growth of most vegetative cells in the filament is temporarily arrested due to nutritional deprivation, but developing heterocysts require intense transcriptional activity. Therefore, the coexistence of arrested vegetative cells and actively developing prospective heterocysts relies on the simultaneous operation of somewhat opposite transcriptional programs. We have identified genes with multiple nitrogen-responsive transcriptional starts appearing in seemingly paradoxical combinations. For instance, sigA, encoding the RNA polymerase housekeeping sigma factor, is transcribed from one major nitrogen stress-repressed promoter and from a second, nitrogen stress-induced promoter. Here we show that both promoters are expressed with complementary temporal dynamics. Using a gfp reporter we also show that transcription from the inducible promoter takes place exclusively in differentiating heterocysts and is already detected before any morphological or fluorescence signature of differentiation is observed. Tandem promoters with opposite dynamics could operate a compensatory mechanism in which repression of transcription from the major promoter operative in vegetative cells is offset by transcription from a new promoter only in developing heterocyst. 4 Introduction Cyanobacteria are considered responsible for the origin of the oxygenic atmosphere on Earth (Lyons et al., 2014). Nowadays, being the most abundant photosynthetic organisms on Earth, cyanobacteria play essential roles in the renewal of atmospheric oxygen and the production of biologically available nitrogen, both as free-living organisms and as partners in nitrogen-fixing symbioses with plants and fungi. Cyanobacteria have a wide ecological distribution that ranges from deserts to all kinds of water reservoirs, suggesting the operation of sophisticated adaptation mechanisms. Filamentous cyanobacteria are developmentally complex prokaryotes that exhibit a variety of cell differentiation processes, including the formation of spore-like cells, motile hormogonia or nitrogen-fixing heterocysts. Heterocystous cyanobacteria are filamentous photosynthetic organisms that grow either as strings of undifferentiated photosynthetic cells (called vegetative) or, upon nitrogen deficiency, as filaments containing vegetative cells and heterocysts, the latter specialized in fixation of atmospheric nitrogen. The differentiation of heterocysts is induced by environmental clues and leads to a multicellular lifestyle in which two cell types with different, complementary metabolic capabilities, cooperate to achieve growth of the filament as a whole (Flores and Herrero, 2010; Muro- Pastor and Hess, 2012). Therefore, when growth is supported by atmospheric nitrogen rather than fixed nitrogen, heterocystous cyanobacteria are obligate multicellular prokaryotes. Transition to a nitrogen-fixing regime involves a period of time in which the growth of the filament transiently stops as a consequence of nutritional deprivation. Such arrest correlates with reduced transcription of 5 several housekeeping genes, including those for RNA polymerase (Flaherty et al., 2011). Once mature heterocysts are fully functional nitrogen fixation begins and growth resumes. Cyanobacterial adaptation to nitrogen deficiency is mainly orchestrated at the transcriptional level by NtcA, a CRP/FNR-family transcription factor that is also required for heterocyst differentiation (Frías et al., 1994; Herrero et al., 2001). The activity of NtcA is modulated by the amount of 2-oxoglutarate (Vázquez- Bermúdez et al., 2002), which reflects the nitrogen status of the cells (Muro- Pastor et al., 2001). In the presence of combined nitrogen, the level of 2- oxoglutarate is low and NtcA has low affinity to target promoters, whereas, under nitrogen depletion, increased levels of 2-oxoglutarate stimulate binding of NtcA to its targets (Zhao et al., 2010). The consensus sequence for NtcA binding, initially described as GTAN8TAC (Luque et al., 1994), has been redefined as GTN10AC after genome-wide analyses involving RNASeq (Mitschke et al., 2011) and ChIP-Seq (Picossi et al., 2014). Depending on the location of the binding motif with respect to transcriptional starts, NtcA can act either as an activator or as a repressor of transcription (Mitschke et al., 2011; Picossi et al., 2014). In addition to NtcA, the developmental response that leads to heterocyst differentiation requires HetR, a regulator specifically involved in cellular differentiation (Buikema and Haselkorn, 1991; Black et al., 1993). In the context of heterocyst differentiation both factors are mutually dependent (Muro- Pastor et al., 2002) and both genes show increased transcription in developing heterocysts (Black et al., 1993; Olmedo-Verd et al., 2006). Overexpression of HetR promotes differentiation, leading to a MCH (multiple contiguous 6 heterocysts) phenotype (Buikema and Haselkorn, 2001). Although purified HetR has been shown to bind inverted repeats upstream of hetP (Higa and Callahan, 2010) or hetZ (Du et al., 2012), it is currently unknown how such DNA-binding activity relates to HetR-mediated regulation of transcription. The morphological and functional transformation of a vegetative cell into a mature heterocyst requires transcription of many genes whose products are exclusively required in the context of heterocyst differentiation and function, such as those involved in synthesis and deposition of the polysaccharide and glycolipid layers of the heterocyst envelope. In addition, the transition to a different growth regime at the expense of molecular nitrogen involves a drastic change in the physiology of the organism as a whole. Because all these changes take place in filaments that contain two cell types (vegetative cells and prospective heterocysts), the transcriptional responses taking place in each of them appear combined in global analyses of the transcriptome. In order to dissect the transcriptional landscape of complex organisms, a procedure called differential RNASeq (dRNASeq) has been applied to the global transcriptional analysis of several bacteria. dRNASeq involves selection of primary transcripts that have a 5’ triphosphate (in contrast to processed RNAs having a 5’ monophosphate) and allows the identification of true transcriptional starts at single base resolution, thus providing an unprecedented view of transcriptional initiation and facilitating direct identification of promoter sequences (Sharma and Vogel, 2014). 7 A dRNASeq-based analysis of the transcriptome of Nostoc sp. strain PCC 7120 allowed the identification of all transcriptional starts along the genome as well as the classification of nitrogen-responsive promoters into different categories according to their dynamics upon nitrogen step down (Mitschke et al., 2011). The transcriptional landscape of this cyanobacterium includes many genes transcribed from complex promoter regions with several transcriptional starts. Upon inspection of the dataset we noticed that some housekeeping genes contained combinations of promoters with opposite dynamics in response to nitrogen deficiency. Here we further analyse the cases of sigA, encoding the housekeeping sigma factor SigA (Brahamsha and Haselkorn, 1991), and asl4317-asl4316, the latter encoding phospho-N-acetylmuramoyl-pentapeptide- transferase (MraY). According to RNASeq data, transcription from the major promoter of sigA is repressed in response to nitrogen deficiency while another promoter is induced (Mitschke et al., 2011). We show here that both promoters operate with complementary temporal (and spatial) dynamics, since the inducible promoter is expressed exclusively in developing heterocysts. Also in the case of asl4317, one nitrogen stress-inducible and one nitrogen stressrepressed promoter are observed. We hypothesize that the presence of such seemingly paradoxical combinations of promoters in housekeeping genes could allow increased expression in prospective heterocyst while vegetative cells are undergoing a generalized reduction of transcription as a consequence of nutritional stress. Results and discussion 8 Nitrogen regulated promoters exhibiting opposite dynamics in Nostoc sp. strain PCC 7120 The dynamics of use of transcriptional starts in response to nitrogen deficiency has been analysed genome-wide in Nostoc sp. strain PCC 7120 and its heterocyst-deficient mutant derivative hetR by applying a dRNASeq-based procedure (Mitschke et al., 2011). The global analysis of the HetR-dependent transcriptome led to the identification of a sequence motif (DIF1, 5’TCCGGA3’, around position -35 of regulated promoters), that appeared in the previously characterized heterocyst-specific promoters of several genes including the small RNA NsiR1 (Ionescu et al., 2010), hetR (Rajagopalan and Callahan, 2010), or sigC (encoding the heterocyst-specific alternative sigma factor SigC) (Brahamsha and Haselkorn, 1992; Aldea et al., 2007). Based on the available dataset of nitrogen-regulated transcriptional start sites, we have now systematically searched for promoter regions containing combinations of regulated promoters that exhibit opposite dynamics in response to nitrogen deficiency (inducible and repressed). Table 1 shows promoter regions that contain at least one nitrogen stress-repressed promoter (DEF- category) and one nitrogen stress-inducible, HetR-dependent, promoter (DIF+ category) (Mitschke et al., 2011). In the case of the DIF+ promoters, the presence of sequences matching the DIF1 motif is also included in Table 1. We decided to further characterize transcription of two genes encoding housekeeping functions, sigA, encoding the RNA polymerase housekeeping sigma factor, and of asl4317-mraY, the latter encoding MraY, involved in peptidoglycan metabolism. In both cases, repression of transcription would be consistent with 9 growth arrest as a consequence of nitrogen deprivation, but the identification of nitrogen stress-induced promoters was however unexpected. Interestingly, both in the case of sigA and of asl4317, the presence of sequences matching a DIF1 motif suggests heterocyst-specific transcription. Nitrogen regulated promoters of sigA Transcription of sigA had been initially analyzed by primer extension, showing transcripts with five different 5’ ends located at -328 (P1), -592 (P2), -605 (P3), - 800 (P4) and -868 (P5) with respect to the translational start. P1 was identified as the major promoter being operative both in the presence or absence of combined nitrogen (Brahamsha and Haselkorn, 1991). The use of dRNASeq later confirmed all of them except P2 (-592) as promoters producing true primary 5’ ends, suggesting the 5’ ends at -592 are the result of processing of longer transcripts (Mitschke et al., 2011). According to dRNASeq data, although transcription from P4 and P5 appears somewhat modulated by nitrogen availability, transcription from P1 and P3 (see a scheme in Fig. 1A) shows the strongest response to nitrogen deprivation. Transcription from P1 is repressed under nitrogen deprivation (log2 fold change= -3.43 at 8 h after nitrogen stepdown), while transcription from P3 is inducible upon nitrogen deprivation in a HetR-dependent manner (log2 fold change= 3.66 at 8 h after nitrogen step down) (Mitschke et al., 2011). Accordingly, P1 was classified as a DEF- transcript, whereas P3 was classified as a DIF+ transcript (Mitschke et al., 2011). 16 This work was supported by grants BFU2013-48282-C2-1 from Ministerio de Economía y Competitividad, and BFU2016-74943-C2-1-P from Agencia Estatal de Investigación (AEI), Ministerio de Economía, Industria y Competitividad, both cofinanced by FEDER. MBA is the recipient of a predoctoral contract from Ministerio de Educación, Cultura y Deportes, Spain (FPU014/05123). REFERENCES Aldea, M.R., Mella-Herrera, R.A., and Golden, J.W. (2007) Sigma factor genes sigC, sigE, and sigG are upregulated in heterocysts of the cyanobacterium Anabaena sp. strain PCC 7120. J Bacteriol 189: 8392-8396. 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. Brahamsha, B., and Haselkorn, R. (1991) Isolation and characterization of the gene encoding the principal sigma factor of the vegetative cell RNA polymerase from the cyanobacterium Anabaena sp. strain PCC 7120. J Bacteriol 173: 2442- 2450. Brahamsha, B., and Haselkorn, R. (1992) Identification of multiple RNA polymerase sigma factor homologs in the cyanobacterium Anabaena sp. strain PCC 7120: cloning, expression, and inactivation of the sigB and sigC genes. J Bacteriol 174: 7273-7282. Brenes-Álvarez, M., Olmedo-Verd, E., Vioque, A., and Muro-Pastor, A.M. (2016) Identification of conserved and potentially regulatory small RNAs in heterocystous cyanobacteria. Front Microbiol 7: 48. 17 Buikema, W.J., and Haselkorn, R. (1991) Characterization of a gene controlling heterocyst differentiation in the cyanobacterium Anabaena 7120. Genes Dev 5: 321-330. Buikema, W.J., and Haselkorn, R. (2001) Expression of the Anabaena hetR gene from a copper-regulated promoter leads to heterocyst differentiation under repressing conditions. Proc Natl Acad Sci USA 98: 2729-2734. Du, Y., Cai, Y., Hou, S., and Xu, X. (2012) Identification of the HetR recognition sequence upstream of hetZ in Anabaena sp. strain PCC 7120. J Bacteriol 194: 2297-2306. Flaherty, B.L., Van Nieuwerburgh, F., Head, S.R., and Golden, J.W. (2011) Directional RNA deep sequencing sheds new light on the transcriptional response of Anabaena sp. strain PCC 7120 to combined-nitrogen deprivation. BMC Genomics 12: 332. Flores, E., and Herrero, A. (2010) Compartmentalized function through cell differentiation in filamentous cyanobacteria. Nat Rev Microbiol 8: 39-50. Frías, J.E., Flores, E., and Herrero, A. (1994) Requirement of the regulatory protein NtcA for the expression of nitrogen assimilation and heterocyst development genes in the cyanobacterium Anabaena sp. PCC 7120. Mol Microbiol 14: 823-832. Herrero, A., Muro-Pastor, A.M., and Flores, E. (2001) Nitrogen control in cyanobacteria. J Bacteriol 183: 411-425. Higa, K.C., and Callahan, S.M. (2010) Ectopic expression of hetP can partially bypass the need for hetR in heterocyst differentiation by Anabaena sp. strain PCC 7120. Mol Microbiol 77: 562-574. 18 Ionescu, D., Voβ, B., Oren, A., Hess, W.R., and Muro-Pastor, A.M. (2010) Heterocyst-specific transcription of NsiR1, a non-coding RNA encoded in a tandem array of direct repeats in cyanobacteria. J Mol Biol 398: 177-188. Lehner, J., Zhang, Y., Berendt, S., Rasse, T.M., Forchhammer, K., and Maldener, I. (2011) The morphogene AmiC2 is pivotal for multicellular development in the cyanobacterium Nostoc punctiforme. Mol Microbiol 79: 1655-1669. Luque, I., Flores, E., and Herrero, A. (1994) Molecular mechanism for the operation of nitrogen control in cyanobacteria. EMBO J 13: 2862-2869. Lyons, T.W., Reinhard, C.T., and Planavsky, N.J. (2014) The rise of oxygen in Earth's early ocean and atmosphere. Nature 506: 307-315. McGrath, P.T., Lee, H., Zhang, L., Iniesta, A.A., Hottes, A.K., Tan, M.H. et al. (2007) High-throughput identification of transcription start sites, conserved promoter motifs and predicted regulons. Nat Biotechnol 25: 584-592. Mitschke, J., Vioque, A., Haas, F., Hess, W.R., and Muro-Pastor, A.M. (2011) Dynamics of transcriptional start site selection during nitrogen stress-induced cell differentiation in Anabaena sp. PCC7120. Proc Natl Acad Sci USA 108: 20130-20135. Muro-Pastor, A.M. (2014) The heterocyst-specific NsiR1 small RNA is an early marker of cell differentiation in cyanobacterial filaments. MBio 5: e01079-01014. Muro-Pastor, A.M., and Hess, W.R. (2012) Heterocyst differentiation: from single mutants to global approaches. Trends Microbiol 20: 548-557. Muro-Pastor, A.M., Valladares, A., Flores, E., and Herrero, A. (1999) The hetC gene is a direct target of the NtcA transcriptional regulator in cyanobacterial heterocyst development. J Bacteriol 181: 6664-6669. 19 Muro-Pastor, A.M., Valladares, A., Flores, E., and Herrero, A. (2002) Mutual dependence of the expression of the cell differentiation regulatory protein HetR and the global nitrogen regulator NtcA during heterocyst development. Mol Microbiol 44: 1377-1385. Muro-Pastor, M.I., Reyes, J.C., and Florencio, F.J. (2001) Cyanobacteria perceive nitrogen status by sensing intracellular 2-oxoglutarate levels. J Biol Chem 276: 38320-38328. Olmedo-Verd, E., Muro-Pastor, A.M., Flores, E., and Herrero, A. (2006) Localized induction of the ntcA regulatory gene in developing heterocysts of Anabaena sp. strain PCC 7120. J Bacteriol 188: 6694-6699. Picossi, S., Flores, E., and Herrero, A. (2014) ChIP analysis unravels an exceptionally wide distribution of DNA binding sites for the NtcA transcription factor in a heterocyst-forming cyanobacterium. BMC Genomics 15: 22. Quereda, J.J., Ortega, A.D., Pucciarelli, M.G., and Garcia-Del Portillo, F. (2014) The Listeria small RNA Rli27 regulates a cell wall protein inside eukaryotic cells by targeting a long 5'-UTR variant. PLoS Genet 10: e1004765. Rajagopalan, R., and Callahan, S.M. (2010) Temporal and spatial regulation of the four transcription start sites of hetR from Anabaena sp. strain PCC 7120. J Bacteriol 192: 1088-1096. Rosario, C.J., and Tan, M. (2016) Regulation of Chlamydia gene expression by tandem promoters with different temporal patterns. J Bacteriol 198: 363-369. Sandh, G., Ramstrom, M., and Stensjö, K. (2014) Analysis of the early heterocyst Cys-proteome in the multicellular cyanobacterium Nostoc punctiforme reveals novel insights into the division of labor within diazotrophic filaments. BMC Genomics 15: 1064. 20 Sharma, C.M., and Vogel, J. (2014) Differential RNA-seq: the approach behind and the biological insight gained. Curr Opin Microbiol 19: 97-105. Vázquez-Bermúdez, M.F., Herrero, A., and Flores, E. (2002) 2-Oxoglutarate increases the binding affinity of the NtcA (nitrogen control) transcription factor for the Synechococcus glnA promoter. FEBS Lett 512: 71-74. Videau, P., Rivers, O.S., Ushijima, B., Oshiro, R.T., Kim, M.J., Philmus, B., and Cozy, L.M. (2016) Mutation of the murC and murB genes impairs heterocyst differentiation in Anabaena sp. Strain PCC 7120. J Bacteriol 198: 1196-1206. Zhao, M.X., Jiang, Y.L., He, Y.X., Chen, Y.F., Teng, Y.B., Chen, Y. et al. (2010) Structural basis for the allosteric control of the global transcription factor NtcA by the nitrogen starvation signal 2-oxoglutarate. Proc Natl Acad Sci U S A 107: 12487-12492. 21 0 Table 1. Genes identified in Nostoc sp. strain PCC 7120 with at least one TSS in the DEF- category and one TSS in the DIF+ category. Reads RNASeqa WT hetR Gene TSSa Classa 0 h 8 h 0 h 8 h DIF1 motifb Annotationc asr1775 2134104f DEF - 104 42 386 16 TACGGA Unknown protein 2134180f DIF + 7 164 1 1 all2736 3333806r DEF - 737 445 493 357 Hypothetical protein 3333817r DIF + 17 519 1 1 asr3342 4038302f DEF - 8614 3003 4732 1953 Acyl carrier protein 4038311f DIF + 1 22 8 0 alr3402 4113609f DIF + 15 289 1 4 Nucleoside diphosphate kinase 4113655f DEF - 87 23 46 26 all3652 4408394r DEF - 430 251 315 150 Phosphoribosylformyl glycinamidine synthetase II 4408666r DIF + 6 26 9 6 asl4317 - mraY 5171243r DEF - 1318 438 1575 344 TGCGGA Hypothetical protein - MraY 5171277r DIF + 47 428 11 7 alr4485 5369226f DEF - 33 4 11 18 ABC transporter, permease protein 5369311f DIF + 5 71 2 1 all4884 5821721r DEF - 37 13 43 14 Hypothetical protein 5821959r DIF + 16 719 3 1 all5263 6279167r (P1) DEF - 1116 145 432 528 TCCGGA Transcription initiation factor; SigA 6279444r (P3) DIF + 2 53 1 1 a Position, classification and number of reads for each TSS are shown according to RNASeq data (Mitschke et al., 2011). b Presence of sequences matching a DIF1 motif (TCCGGA) (Mitschke et al., 2011). c According to Cyanobase (http://genome.microbedb.jp/cyanobase) 22 FIGURE LEGENDS Fig. 1. Nitrogen-regulated promoters P1 and P3 of the sigA gene. A. Scheme of the promoter region of sigA showing the positions of two nitrogenregulated transcriptional starts, located at positions -605 (P1) and -328 (P3) with respect to the translational start (genomic coordinates 6279167r and 6279444r, respectively). B. Sequences around the nitrogen stress-repressed transcriptional starts of sigA (bold, underlined, and marked with a bent arrow), gifA and rbcL (bold, underlined) are aligned with respect to their -10 boxes shown in grey. Putative NtcA binding sites are shown underlined with the nucleotides matching the palindromic consensus GTAN8TAC shown in red. C. Primer extension analysis of transcripts from P1 in RNA isolated from the wild type strain and the ntcA derivative CSE2 (Frías et al., 1994), after the cells were transferred to nitrogen-free medium for the number of hours indicated in each case. D. Electrophoretic mobility shift assays with purified NtcA and the DNA fragment comprising P1 (positions -484 to -227 with respect to the translational start of sigA), P32-labelled using -dATP and T4 polynucleotide kinase. The empty triangle points to the free fragment whereas the filled triangle indicates the position of the retarded fragment. E. Sequences upstream of the nitrogen stress-induced transcriptional start of sigA (bold, underlined, and marked with a bent arrow), are shown together with those upstream the heterocyst-specific transcriptional starts of NsiR1, hetR and sigC (bold, underlined). Sequences matching the consensus for the DIF1 motif TCCGGA are shown in red. F. Primer extension analysis of transcripts from P3 in RNA isolated from the wild type strain (WT) or the hetR derivative 216 (Buikema and Haselkorn, 1991) 23 after the cells were transferred to nitrogen-free medium for the number of hours indicated in each case. Nitrogen step-down, RNA isolation, primer extension and EMSA with a purified his-tagged version of NtcA were carried out as previously described (Muro-Pastor et al., 1999). All oligonucleotides used in this work are shown in Supplemental Table S1. Fig. 2. Expression of PsigA-gfp along nitrogen fixing filaments of Nostoc sp. strain PCC 7120 bearing pSAM330. A. Schematic representation of the location of the transcriptional starts corresponding to P1 and P3 (bent arrows) and the P3 promoter segment transcriptionally fused to gfpmut2 (bearing its ribosome binding site) in plasmid pSAM330, a derivative of pCSEL24 that is integrated in the alpha megaplasmid of Nostoc sp. strain PCC 7120 as described previously (Olmedo-Verd et al., 2006). B. Confocal fluorescence image of a filament growing on top of nitrogen-free medium is shown for the red and green channels. A region of the filament is enhanced for better observation of green fluorescence and shown as an inset on the left side. Fluorescence was analysed and quantified using a Leica TCS SP2 confocal laser-scanning microscope. GFP and red fluorescence were imaged using the 488 nm line supplied by an argon ion laser. Fluorescent emissions were monitored by collection across windows of 500-538 nm (GFP imaging) and 630-700 nm (chlorophyll fluorescence). Quantification of the signals along the filament is shown separately for the red (C) and green (D) channels. Mature heterocysts (numbers 1, 2, 3, 5, 6, 8 and 9) are indicated in white (B) or black (D). Immature heterocysts (numbers 4 and 7) are indicated in red. Prospective heterocysts are indicated with single asterisks. Black arrows indicate the positions of mature 24 heterocysts, whereas red arrows indicate immature heterocyst. The segment of the graphs corresponding to the inset in B is framed with a black rectangle. Images were treated with ImageJ 1.45s software (Rasband, W.S., ImageJ, U. S. National Institutes of Health, Bethesda, Maryland, USA, http://imagej.nih.gov/ij/, 1997-2016). Fig. 3. Nitrogen-regulated promoters of asl4317-all4316 (mraY). A. Scheme of the two genes and the promoter region showing the positions of the two nitrogen regulated transcriptional starts, located at positions -39 and -73 with respect to the translational start. B. Primer extension analysis of transcripts starting at position -39 in RNA isolated from the wild type strain (WT) or the ntcA mutant strain CSE2 after the cells were transferred to nitrogen-free medium for the number of hours indicated in each case. C. Sequences upstream of the nitrogen stress-induced transcriptional start of asl4317 (bold, underlined, and marked with a bent arrow), are shown together with those upstream the heterocyst-specific transcriptional starts of sigA, NsiR1, hetR and sigC (bold, underlined). Sequences matching the consensus for the DIF1 motif are shown in red. D. Primer extension analysis of transcripts starting at position -73 in RNA isolated from the wild type strain (WT) or the hetR mutant strain 216 after the cells were transferred to nitrogen-free medium for the number of hours indicated in each case. Fig. 4. Schematic representation of the combined operation of P1 and P3 promoters of the sigA gene upon nitrogen step down. In the presence of combined nitrogen P1 is operative in vegetative cells (thick grey arrow under 25 sigA), while transcription from P3 does not take place. Upon nitrogen step down leading to increased levels of 2-oxoglutarate (6-12 h), transcription from the major P1 promoter is repressed by NtcA in all cells (thin grey arrow under sigA) while P3 is induced only in prospective heterocysts (thick red arrow under sigA). Because the amount of NtcA is higher in developing heterocysts, repression of P1 is expected to be stronger in those cells. Once the heterocysts are mature (20-24 h) the transcription from P3 is reduced (thin red arrow under sigA). Concerning P1 we can only speculate that repression by NtcA is alleviated as the transient limitation in nitrogen availability is reverted by fixation of atmospheric nitrogen. Similarly, expression from P3 is highest during early stages of development but is reduced as the heterocysts become mature. The result of such combination is that, while there is a transient reduction in expression in vegetative cells, expression remains somewhat constant in developing heterocysts that are actively differentiating. [2-OG], concentration of 2-oxoglutarate.