N and C control of ABC-type bicarbonate transporter Cmp and its LysR-type transcriptional regulator CmpR in a heterocyst-forming cyanobacterium, Anabaena sp.

Depósito de investigación de la Universidad de Sevilla https://idus.us.es/ “This is an Accepted Manuscript of an article published in [INTERNATIONAL JOURNAL OF SYSTEMATIC AND EVOLUTIONARY MICROBIOLOGY] on [July 2018], available at: https://doi.org/[10.1099/ijsem.0.002897 ].” N and C control of ABC-type bicarbonate transporter Cmp and its LysR-type transcriptional regulator CmpR in a heterocyst-forming cyanobacterium, Anabaena sp.emi_ 2683 1..151..15 Rocío López-Igual, Silvia Picossi, Javier López-Garrido, Enrique Flores and Antonia Herrero* Instituto de Bioquímica Vegetal y Fotosíntesis, Consejo Superior de Investigaciones Científicas and Universidad de Sevilla, Américo Vespucio 49, E-41092 Seville, Spain. Summary In the model, heterocyst-forming cyanobacterium Anabaena sp. PCC 7120, gene cluster alr2877-alr2880, which encodes an ABC-type transport system, was induced under conditions of carbon limitation and its inactivation impaired the uptake of bicarbonate. Thus, this gene cluster encodes a Cmp bicarbonate transporter. ORF all0862, encoding a LysR-type transcriptional regulator, was expressed under carbon limitation and at higher levels in the absence than in the presence of combined nitrogen, with a positive effect of the N-control transcription factor NtcA. all0862 was expressed from two putative transcription start sites located 164 and 64 bp upstream from the gene respectively. The latter was induced under carbon limitation and was dependent on positive autoregulation by All0862. All0862 was required for the induction of the Cmp bicarbonate transporter, thus representing a CmpR regulator of Anabaena sp. These results show a novel mode of co-regulation by C and N availability through the concerted action of Nand C-responsive transcription factors. Introduction Ecologically, cyanobacteria are relevant microorganisms that are responsible for an important fraction of the primary productivity in our planet. They are important C and N fixers that impact the cycling of CO2at a global scale and, through the fixation of atmospheric N2, contribute to replenish manageable N into the Biosphere. Cyanobacteria fix CO2through the enzyme ribulose-1,5bisphosphate carboxylase/oxygenase (Rubisco), which has a low affinity for CO2and, moreover, also accepts O2 as a substrate. To increase the efficiency of CO2fixation, cyanobacteria have developed a characteristic carbon concentrating mechanism (CCM) constituted by CO2and HCO3-transporters, carbonic anhydrases, and a proteinaceous compartment, the carboxysome, where the cellular Rubisco is confined (Price et al., 2008). Through this system, ambient CO2and HCO3-are concentrated in the cytoplasm of the cyanobacterial cell in the form of HCO3-, which then enters the carboxysome where it is converted to CO2that accumulates at a high enough concentration to allow an efficient operation of Rubisco. Concerning HCO3-transporters, two types of Na+- dependent systems (BicA and SbtA) and one ABC-type system (denoted Cmp or BCT1) have been identified in different cyanobacteria (Kaplan et al., 2008; Price, 2011). The high affinity transporter Cmp, encoded by the cmp operon, has been identified and characterized in two unicellular cyanobacteria: Synechococcus sp. PCC 7942 (Omata et al., 1999; Maeda et al., 2000) and Synechocystis sp. PCC 6803 (Omata et al., 2001), although homologues of this system are present in the sequenced genomes of at least other eight different cyanobacteria, including Anabaena sp. PCC 7120 (see Price et al., 2008). Concerning N assimilation, cyanobacteria mainly use inorganic compounds such as nitrate, ammonium or urea, and many cyanobacteria are able to fix atmospheric N. In cyanobacteria, N control is mediated by NtcA, a member of the CRP family of bacterial transcriptional regulators (Herrero et al., 2001). NtcA together with 2-oxoglutarate (2-OG), a metabolic signal of N deficiency, binds to specific DNA sites with the sequence signature GTAN8TAC and activates or represses the expression of multiple target genes (Luque et al., 1994; Herrero et al., 2001; Valladares et al., 2008). To fix N2under oxic conditions, some filamentous cyanobacteria produce specialized cells called heterocysts that do not perform oxygenic photosynthesis (Wolk et al., 1994). The specific program of gene expression underlying heterocyst differentiation is integrated into the suite of the organisms’ responses to Received 16 September, 2011; revised 22 November, 2011; accepted 25 November, 2011. *For correspondence. E-mail herrero@ ibvf.csic.es; Tel. (+34) 95 448 9522; Fax (+34) 95 446 0065. emi_2683 Environmental Microbiology (2011) doi:10.1111/j.1462-2920.2011.02683.x © 2011 Society for Applied Microbiology and Blackwell Publishing Ltd 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 nitrogen deprivation that is orchestrated by NtcA (Herrero et al., 2004; Flores and Herrero, 2010). LysR-type transcriptional regulators (LTTR) represent the largest known family of bacterial DNA-binding factors involved in regulation in the context of a wide range of biological functions that include metabolism, cell division, quorum sensing, virulence, motility, nitrogen fixation, oxidative stress, toxin production, attachment and secretion (Maddocks and Oyston, 2008). LTTRs have a conserved structure, exhibiting a helix – turn – helix, DNA-binding motif at the N-terminus, and a C-terminal cofactor-binding domain. They can act as activators or repressors, and their function frequently depends on a co-inducer molecule (e.g. Picossi et al., 2007). A generally accepted LTTR box in the form T-N11-A is usually present in the repressor-, but not in the activator-, DNAbinding sites (e.g. Parsek et al., 1994). Additionally, the apo-form and co-inducer-bound LTTR may differ in their affinity for the DNA, which may affect its preference for activator or repressor sites (Maddocks and Oyston, 2008). Among the LTTR factors identified in cyanobacteria, in Synechocystis sp. PCC 6803 expression of the cmp operon is activated by CmpR, a LTTR encoded by ORF sll0030, which is located immediately upstream from cmpA and transcribed divergently from it, whereas in Synechococcus sp. PCC 7942, CmpR is unlinked to the cmp operon (Omata et al., 2001). A different LTTR, named as CcmR or NdhR, has been described to negatively regulate, besides its own gene and other genes, the ndh3 operon encoding a CO2transporter and the sbtA/sbtB genes encoding a HCO3-transporter in Synechocystis sp. PCC 6803 (Figge et al., 2001; Wang et al., 2004), as well as the bicA operon encoding a HCO3-transporter in Synechococcus sp. PCC 7002 (Woodger et al., 2007). In the genomic sequence of the filamentous, heterocyst-forming model cyanobacterium Anabaena sp. PCC 7120 (Kaneko et al., 2001), seven homologues of LTTRs can be identified. For only one of these factors, namely ORF all0602 encoding the NtcB protein involved in the regulation of expression of the nirA operon, molecular targets have been experimentally characterized (Frías et al., 2000). In this work we have experimentally determined that gene cluster alr2877-alr2880 encodes the ABC-type Cmp (BCT1) HCO3-transport system of Anabaena sp. PCC 7120, which to the best of our knowledge represents the first Ci transporter identified in a heterocyst-forming cyanobacterium. Expression of this system responds primarily to the Ci regime as regulated by LTTR All0862, which itself responds to the carbon regime through positive autoregulation and to the N regime through NtcAmediated activation. Results In an attempt to identify the role of ABC-transport systems of heterocyst-forming cyanobacteria, we studied the gene cluster alr2877-alr2880 of Anabaena sp. PCC 7120, which in the CyanoBase databank (Kaneko et al., 2001) (Fig. 1A) is annotated as corresponding to the ABC-type bicarbonate transport system Cmp characterized in the unicellular cyanobacteria Synechocystis sp. PCC 6803 and Synechococcus sp. PCC 7942 (Omata et al., 1999; 2001; Maeda et al., 2000). Expression of alr2877-alr2880 Expression of the strain PCC 7120 gene cluster alr2877alr2880 and the influence on expression of the C and N regime were studied by Northern blot analysis with an alr2877 probe. RNA was extracted from whole filaments grown with ammonium under a high Ci regime (liquid medium supplemented with 10 mM HCO3-and bubbled with air enriched with CO2), and incubated under a low Ci regime (liquid medium without HCO3-and bubbled with air) in media containing ammonium or no combined nitrogen (Fig. 1B). Hybridization signals with an upper size limit corresponding to c. 5.4 kb were detected. Thus, these transcripts would also include the messages of the alr2878, alr2879 and alr2880 genes (together 5375 bp). Under high C regime, expression of this gene cluster was negligible, and it increased highly upon incubation under low C regime, both in the presence of ammonium and in the absence of combined N. Thus, expression of this gene cluster responds mainly to the C supply. Inactivation of alr2877 To investigate the role of the Anabaena gene cluster alr2877-alr2880, we sought the generation of mutant strains with inactivated versions of this putative transport system. Strains CSJL1 and CSJL2 bear, respectively, the SmrSpr-encoding plasmid pCSJL5 and the Kmr-encoding plasmid pCSJL6 inserted into ORF alr2877 in all chromosome copies (see Experimental procedures and Fig. S1 for details). The HCO3-transport activity was assayed in filaments of strains CSJL1 and CSJL2 grown under a high Ci regime and incubated under a low Ci regime in comparison with the wild type. Data presented in Fig. 2 show that in the two mutant strains HCO3-transport activity was impaired, with values of c. 0.6 that of the wild type. On the other hand, strains CSJL1 and CSJL2 were not impaired in nitrate or nitrite uptake (not shown). These results indicate that alr2877 is involved in bicarbonate transport in strain PCC 7120. Because of the high similarity of the 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 emi_2683 2R. López-Igual et al. © 2011 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology Alr2877-Alr2878-Alr2879-Alr2880 gene products to the components of the bicarbonate Cmp transport systems of Synechococcus and Synechocystis, we identified the alr2877-alr2880 gene cluster as the cmp operon of strain PCC 7120. Under C limitation (shaken BG11 cultures), growth rate of strains CSJL1 and CSJL2 was 0.89 and 0.72 (media of two experiments with similar results), respectively, that of the wild type. Regulation of the expression of the Anabaena cmp operon by NtcA To gain insight into the regulation of the cmp operon, we studied the expression of this operon in different mutants derived from strain PCC 7120 and impaired in regulatory elements. First we used strain CSE2, a mutant lacking the NtcA transcription factor involved in N control (Frías et al., 1994). Northern blot analysis showed that upon transfer of ammonium-grown cells to conditions of C limitation in the presence of ammonium or in the absence of combined nitrogen, induction of the cmp operon took place in strain CSE2 (Fig. 1C). However, as N deprivation persisted, expression of cmp in strain CSE2 became lower than in the wild type (compare Fig. 1C with 1B). To analyse the regulation of the expression of the cmp operon, primer extension analysis was performed using two different oligonucleotide primers: alr2877-5 (Fig. 3A) and alr2877-6 (not shown). A single 5′RNA end was detected that was located at nucleotide position -220 with regard to the start of alr2877 (Fig. 3A, PCC 7120). This RNA was observed only after incubation under C limitation. When a DNA fragment encompassing sequences around this position was used in DNase I footprinting assays with purified NtcA protein, clear changes in the digestion pattern due to the presence of NtcA that included a protected region and some hypersensitive positions indicative of direct binding of the protein were observed (Fig. 3B). NtcA-binding sites in DNA have the sequence signature GTAN8TAC (Luque et al., 1994; Herrero et al., 2001; Vázquez-Bermúdez et al., 2002). The DNA region protected by NtcA includes the sequence GTACACTAAATTTC (Fig. 3C), which represents a putative NtcA-binding site. Fig. 1. Northern blot analysis of the expression of alr2877. A. The strain PCC 7120 genomic region of gene cluster alr2877-alr2880 (Kaneko et al., 2001) is depicted. B. RNA was isolated from cultures of strain PCC 7120 (B) or CSE2 (C) grown in media BG110C+NH4+bubbled with a mixture of air and 3% CO2(0) and incubated in medium BG110+NH4+(NH4+) or BG110(N2) bubbled with air for the times indicated in hours. RNA was electrophoresed and hybridized with the probe of alr2877 indicated in (A) (black bar), which was generated by PCR (see Experimental procedures). Hybridization with an rnpB gene probe was used as a loading and transfer control. The size of the largest transcript detected is indicated on the right. (B) and (C) show images of different parts of the same filter. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 emi_2683 Bicarbonate transport regulation in Anabaena 3 © 2011 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology All0862, a carbonand nitrogen-responsive LTTR of Anabaena In a global approach to study the role of the LTTRs of Anabaena sp. PCC 7120, we analysed ORF all0862, which is annotated as encoding an RbcR transcriptional regulator. all0862 is positioned downstream, separated by 462 bp, from a cluster of putative ccm genes (all0868all0863; Kaneko et al., 2001) (Fig. 4A). Expression of all0862 as influenced by the C and N regime was studied by Northern blot analysis with RNA isolated from filaments grown with ammonium under a high Ci regime and transferred to a low Ci regime in the presence of ammonium or in the absence of combined nitrogen. A transcript of c. 1 kb, which would correspond to a monocistronic message of all0862 (918 bp) was detected. Abundance of this transcript was very low under high C conditions, and increased in response to C limitation, both in the presence and absence of combined nitrogen, although induction was stronger in the absence of ammonium (Fig. 4B). On the other hand, induction of all0862 upon C limitation in the absence of ammonium was lower in the ntcA mutant than in the wild type (compare Fig. 4C with 4B). Primer extension experiments using two different oligonucleotide primers, all0863-2 (Fig. 5A) and all0862-9 (not shown), showed the presence of two 5′RNA ends, which were located at nucleotide positions -64 and -164 with respect to the all0862 ORF start. Whereas no significant change in the abundance of the -164 5′RNA end was observed, abundance of the -64 end increased upon a shift from high Ci to low Ci regime (Fig. 5A, PCC 7120). Because induction of all0862 was impaired in the ntcA mutant strain (Fig. 4), direct binding of purified NtcA protein to the promoter region of all0862 was tested. In EMSA assays NtcA, especially in the presence of 2-OG, promoted the appearance of three retarded bands that could correspond to complexes of NtcA bound to at least two DNA sites (Fig. 6B). When two subsets of the former DNA fragment were used, retarded bands promoted by the presence of NtcA were detected in every case (Fig. 6C, E, F). The observed retardation pattern would be consistent with binding of NtcA to at least one site contained in the all0863-2/all0862-6 fragment (fragment F2) and to another one in the HphI/all0862-5 fragment (F3). The faint slower band produced with the all0863-2/ all0862-6 fragment (Fig. 6C and E, grey arrows) could result from a weak interaction of NtcA in an additional site in this fragment. Because in all cases band retardation was competed by the addition of an unrelated DNA fragment including a canonical NtcA-binding site (a PglnA fragment; Frías et al., 1994) but not by a fragment lacking NtcA-binding sites (Fig. 6D–F), interactions of NtcA in the all0862 promoter region appear to be specific. Fig. 2. Uptake of HCO3-in strains PCC 7120, CSJL1, CSJL2 and CSRL2. Time-course of H14CO3-uptake in filaments of strains PCC 7120 (circles), CSJL1 (squares), CSJL2 (triangles) and CSRL2 (crosses) grown in medium BG110C+NH4+bubbled with a mixture of air and 2% CO2(high Ci) and incubated in medium BG110 bubbled with air (low Ci) for 6 h was performed as described in Experimental procedures. Data are the mean and standard deviation of the mean of four independent experiments. Fig. 3. Analysis of the alr2877 gene promoter region. A. Primer extension analysis of the alr2877 gene promoter region in strains PCC 7120 and CSRL2 carried out with primer alr2877-5 and RNA isolated from cultures of strain PCC 7120 or CSRL2 grown in medium BG110C+NH4+bubbled with a mixture of air and 3% CO2(0) and incubated in medium BG110+NH4+bubbled with air for the times indicated in hours. The position of the -220 5′RNA end is indicated. B. NtcA DNase I footprinting on the promoter region of the alr2877 gene carried out with purified NtcA: 0 (1 and 4), 0.8 (2) or 2.4 (3) mM, in the presence of 0.6 mM 2-OG and a DNA fragment (7 fmol) of the alr2877 promoter region amplified by PCR using oligonucleotides alr2877-10 (32P-labelled, upper strand) and alr2877-8 (32P-labelled, lower strand). M, ‘20 bp Molecular Ruler DNA Size Standard’ (Bio-Rad). Vertical lines indicate NtcA footprints. Arrowheads point to hypersensitive sites outside the footprinted windows. C. DNA sequences upstream from alr2877. Overline and underline indicate NtcA protected regions in the upper and lower strand, respectively (for simplicity, only the nucleotide sequence of the upper strand is shown). Arrowheads point to positions of hypersensitivity outside the footprinted windows. T included in an oval indicates the 5′RNA end identified at position -220, shadowed sequences indicate putative -35 and -10 promoter determinants. GTA and TTC in boldface identify the NtcA-binding site (see the text). The positions of the oligonucleotide primers used are indicated with dashed lines. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 emi_2683 4R. López-Igual et al. © 2011 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology emi_2683 Bicarbonate transport regulation in Anabaena 5 © 2011 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology Binding of NtcA to the promoter region of all0862 was also studied by means of DNase I footprinting assays. Using two different DNA fragments of the all0862 promoter region, NtcA promoted changes in the digestion pattern producing two clear windows of protection (Fig. 5B and C). The two DNA regions protected against DNase I digestion include the sequences GTT TTCAAAACTAC and GTATTCAGTGATAC, respectively (Fig. 5D), which represent putative NtcA-binding sites. Additionally, several positions exhibiting enhanced or lowered DNase I sensitivity were detected between the two protected regions. This suggests distortion of the DNA between these regions. All0862 regulates the expression of the cmp, rbc and ccm operons, and is positively autoregulated To understand the role of the All0862 LTTR in Anabaena sp. PCC 7120, mutation of all0862 was undertaken. Strain CSRL2 bears in all chromosome copies a version of all0862 lacking 667 internal bp (see Experimental procedures and Fig. S2 for details). Primer extension analysis of the promoter of the all0862 gene was carried out using RNA from the CSRL2 mutant. Of the two 5′ RNA ends detected in the wild type, only the one corresponding to position -164 (but not that corresponding to position -64) was detected in the mutant (Fig. 5A). Fig. 4. Northern blot analysis of the expression of all0862. A. The strain PCC 7120 genomic region of all0862 (Kaneko et al., 2001) is depicted. RNA was isolated from cultures of strain PCC 7120 (B) or CSE2 (C) grown in medium BG110C+NH4+bubbled with a mixture of air and 3% CO2(0) and incubated in medium BG110+NH4+(NH4+) or BG110(N2) bubbled with air for the times indicated in hours. RNA was electrophoresed and hybridized with the probe of all0862 indicated in (A) (black bar), which was generated by PCR (see Experimental procedures). Hybridization with an rnpB gene probe was used as a loading and transfer control. The approximate size of the transcript detected is indicated at right. (B) and (C) show images of different parts of the same filter. Fig. 5. Analysis of the all0862 gene promoter region. A. Primer extension analysis of the all0862 gene promoter region in strains PCC 7120 and CSRL2 was carried out with primer all0863-2 and RNA isolated from cultures of strain PCC 7120 or CSRL2 grown in medium BG110C+NH4+bubbled with a mixture of air and 3% CO2(0) and incubated in medium BG110+NH4+bubbled with air for the times indicated in hours. The positions of the -64 and -164 5′RNA ends are indicated. B, C. NtcA DNase I footprinting on the promoter region of the all0862 gene carried out with purified NtcA: 0 (1 and 4), 0.8 (2) or 2.4 (3) mMin the presence of 0.6 mM 2-OG and a DNA fragment (7 fmol) of the all0862 promoter region amplified by PCR using oligonucleotides all0863-2 (32P-labelled, lower strand) and all0862-5 (32P-labelled, upper strand) (fragment F1 in Fig. 6) (B) or all0862-6 (32P-labelled, upper strand) and all0863-2 (unlabelled) (fragment F2 in Fig. 6) (C). M, ‘20 bp Molecular Ruler DNA Size Standard’ (Bio-Rad). Vertical lines indicate NtcA footprints Arrowheads point to hypersensitive sites outside the footprinted windows. D. DNA sequences upstream of all0862. Overline and underline indicate NtcA protected regions in the upper and lower strand, respectively (for simplicity, only the nucleotide sequence of the upper strand is shown). Arrowheads point to positions of hypersensitivity outside the footprinted windows. A and C included in ovals indicate the 5′RNA ends identified at position -64 and -164, respectively, and shadowed sequences indicate a putative -10 promoter determinant. GTT/TAC and GTA/TAC in boldface identify NtcA-binding sites (see the text). The positions of the oligonucleotide primers used are indicated with dashed lines. The HphI cutting site used in Fig. 6 is also indicated. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 emi_2683 6R. López-Igual et al. © 2011 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology emi_2683 Bicarbonate transport regulation in Anabaena 7 © 2011 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology These results suggest positive autoregulation of the gene. On the other hand, expression of the cmp operon encoding the Cmp HCO3-transporter, identified above, was studied in strain CSRL2 in comparison with the wild type. Northern analysis showed that the activation of the operon in response to C limitation was greatly impaired in the mutant (Fig. 7A). Strain CSRL2 was complemented by integration into the chromosome of plasmid pCSS144, which includes a wild-type copy of the all0862 gene, producing strain CSS53 (see Experimental procedures for details). In strain CSS53 expression and induction of the cmp operon in response to C limitation was similar to that of the wild type (Fig. 7A). Thus, impaired expression of cmp in mutant strain CSRL2 results from inactivation of all0862. Primer extension experiments indicated that in response to C limitation, the 5′RNA end upstream from cmpA could be detected in the CSRL2 mutant, albeit at levels considerably lower than in the wild type (Fig. 3A). Consistent with results of experiments analysing expression levels of the cmp operon, the activity of HCO3-transport in the CSRL2 mutant was c. 0.6 that of the wild type, which is similar to the values exhibited by the cmp mutant strains CSJL1 and CSJL2 (Fig. 2). Thus, All0862 is required for activation of the cmp operon in response to C limitation. Finally, expression of the rbcLXS operon, encoding Rubisco, and of the all0864 gene, included in cluster all0868-all0863 annotated as encoding components of the carbon dioxide-concentrating mechanism (Kaneko et al., 2001), were studied in cells of strain CSRL2 grown under a high Ci regime and incubated under C limitation, Fig. 6. EMSA assay of the binding of NtcA to the all0862 upstream region. A. The all0862-all0863 intergenic region is depicted, indicating the location of the three DNA fragments used in EMSA assays and the oligonucleotide primers or HphI restriction site used to generate those fragments (see Fig. 5D). B, C. EMSA carried out with purified NtcA (0, 0.26, 0.52, 0.78, 1.5 mM) in the presence or absence of 0.6 mM 2-OG and DNA fragments generated with primers all0863-2 and all0862-5 (fragment F1; 1 fmol, B) or all0863-2 and all0862-6 (fragment F2; 2 fmol, C). D–F. EMSA carried out with purified NtcA [16 nM of a protein preparation different from that used in (B) and (C)], 0.6 mM 2-OG and 32P-labelled fragment F1 (1 fmol), F2 (2 fmol) or F3 (a fragment generated with HphI restriction and primer all0862-5; 2 fmol), in the absence (-) or presence of an excess of 100 or 500 times of the same unlabelled fragment or of an unlabelled fragment of the promoter region of the PCC 7120 glnA gene [generated by PCR with plasmid pAN503 (Tumer et al., 1983) as template and primers GA3 and GA13] or of the Amaranthus hybridus psbA gene [generated with plasmid pRL278 (Black et al., 1993) and primers CK3-1 and CK3-2], as indicated. 0, control with no NtcA. Open arrowheads indicate the positions of the free DNA fragments; closed arrowheads indicate those of putative NtcA – DNA complexes. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 emi_2683 8R. López-Igual et al. © 2011 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology Toppan Best-set Premedia Limited Journal Code: EMI Proofreader: Jason Article No: 2683 Delivery date: 12 December 2011 Page Extent: 14 Copyeditor: Harry AUTHOR QUERY FORM Dear Author, During the preparation of your manuscript for publication, the questions listed below have arisen. Please attend to these matters and return this form with your proof. Many thanks for your assistance. 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