NtcA is responsible for accumulation of the small isoform of ferredoxin:NADP oxidoreductase

++ ++ MM i i c c r r o o b b i i o o l l o o g g y y 0-mic.sgmjournals.org.fama.us.es PP u u b b l l i i ss h h ee d d o o n n l l i i n n ee aa h h ee aa d d oo f f pp r r i i n n t t JJaannuuaarryy 2244,, 22001144,, ddooii:: 11 00 . . 11 00 99 99 // mm i i cc . . 00 . . 00 77 66 00 44 22 -- 00 Microbiology AApprriill 22001144 vol. 160 no. Pt 4 77 88 99 -- 77 99 44 NtcA is responsible for accumulation of the small isoform of ferredoxin:NADP oxidoreductase AA mm i i nn OO mm aa i i r r i i -- NN aa s s s s e e r r ,,†‡,, CC aa r r l l aa VV . . GG a a l l m m oo zz zz i i ,,†§,, AA m m e e l l L L a a t t i i f f i i ,, MM . . I I s s a a b b e e l l MM u u r r o o - - P P a a s s t t o o r r aanndd GG hh a a dd a a AA j j l l a a nn i i Author Affiliations Author Notes CC oo r r r r ee s s pp oo nn dd ee nn c c ee M. Isabel Muro-Pastor [email protected] Ghada Ajlani [email protected] Received 13 December 2013. Accepted 22 January 2014. Abstract In several cyanobacteria, petH , the gene encoding ferredoxin:NADP oxidoreductase (FNR), is transcribed from at least two promoters depending on growth conditions. Two transcripts (short and long) are translated from two different translation initiation sites, resulting in two isoforms (large and small, respectively). Here, we show that in Synechocystis PCC6803 the global transcriptional regulator NtcA activates transcription from the distal petH promoter. Modification of the NtcA-binding site prevents NtcA binding to the promoter in vitro and abolishes accumulation of the small isoform of FNR in vivo . We also demonstrate that a similar petH transcription and translation regime occurs in other cyanobacteria. The conditions under which this system operates provide hints for the function of each FNR isoform. ↵† These authors contributed equally to this work. EE d d i i t t ee d d b b yy : : C.-C. Zhang Introduction The gene petH encodes ferredoxin : NADP oxidoreductase (FNR), an enzyme that is involved in the last step of oxygenic photosynthesis, in cyanobacteria as well as in chloroplasts. FNR provides NADPH for anabolic reactions and is also implicated in the oxidation of NADPH, produced by catabolism or accumulated due to an imbalance in photosynthetic reactions. Most phycobilisomecontaining cyanobacteria possess a large FNR isoform (FNRL) that contains a phycobilisome-linker domain. Fewer strains accumulate a small isoform (FNRS), lacking the linker domain, in addition to FNRL or as the only FNR form (Thomas et al. , 2006). We showed recently that in Synechocystis sp. PCC6803 (hereafter Synechocystis ), following nitrogen starvation, petH produces a transcript bearing a 5′-untranslated region that is longer than the one transcribed under standard conditions. The longer transcript folds into a secondary structure that inhibits FNRL translation initiation and promotes that of FNRS (Omairi-Nasser et al. , 2011). Although FNR isoform accumulation was not examined in the cyanobacterium Anabaena sp. strain PCC7120 (hereafter Anabaena ), its transcript organization is similar to that of Synechocystis . Two transcripts were found for the Anabaena petH ; the shorter is constitutive, whilst the longer is NtcA-dependent and mainly located in heterocysts (Valladares et al. , 1999). NtcA is a global transcriptional regulator that belongs to the cAMP receptor protein (Crp)/Fnr bacterial superfamily, and regulates nitrogen and carbon assimilation genes in cyanobacteria in response to 2-oxoglutarate. 2-Oxoglutarate is an intermediate of the TCA cycle that provides the carbon skeleton for nitrogen incorporation into amino acids. In cyanobacteria, 2-oxoglutarate also functions as a regulatory effector for NtcA and PII, whose activities alter gene expression and metabolism (Muro-Pastor et al. , 2001; Körner et al. , 2003; Osanai et al. , 2006; Luque & Forchhammer, 2008). In many cases, NtcA-activated promoters are similar to class II Crp-dependent promoters, in which the transcription factor binds to a consensus sequence centred at ~40 bases upstream from the transcription start point (Luque & Forchhammer, 2008). A consensus sequence for NtcA binding has been defined as GTAN8TAC (Herrero et al. , 2001; Mitschke et al. , 2011b). The recently solved 1 2 3 2 1 NtcA structure shows that NtcA is a dimeric protein with a very similar overall structure to that of Crp (Llácer et al. , 2010; Zhao et al. , 2010). Putative NtcA-binding sites are present upstream from the promoter that produces the longer petH transcript in Synechocystis . We identified the actual binding site, centred at −41.5 bases from the distal petH transcription start point, and showed that NtcA is required for FNRS synthesis in Synechocystis . Moreover, the petH transcription and translation mechanisms, leading to the accumulation of two FNR isoforms, are conserved in Anabaena and in Synechococcus sp. PCC7002. An NtcA-binding site was identified upstream from the transcription start point producing a longer petH transcript in Anabaena (Valladares et al. , 1999). Here, we show that NtcA binding to this distal promoter is responsible for FNRS translation in heterocysts. Methods Strains and growth conditions. WT and mutants of Synechocystis were grown photoautotrophically, at 33 °C, in a CO2-enriched atmosphere and under continuous light (50 µE m s ), which are standard growth conditions in our laboratory. The medium composition is described in Ughy & Ajlani (2004). For nitrogen starvation, cells were harvested by centrifugation and resuspended in a medium where NaCl replaced NaNO3. When appropriate, media were supplemented with 5 µg streptomycin ml , 50 µg spectinomycin ml or 50 µg kanamycin ml . Synechococcus sp. PCC7002 was grown under the same conditions used for Synechocystis except that A+ medium was used (Stevens & Porter, 1980). Anabaena sp. PCC7120 WT and CSE2 strains were grown photoautotrophically, at 30 °C, in BG110C, which is BG11 medium without NaNO3 and with 10 mM NaHCO3, supplemented with 6 mM NH4Cl plus 12 mM TES (pH 7.5) and bubbled with 1 % CO2 in air until the exponential phase. Then the ammonium was eliminated and the cells incubated for the time indicated in BG110C (nitrogen free), with 1 % CO2 in air. In the case of the CSE2 strain, the medium was supplemented with 2 µg streptomycin ml and 2 µg spectinomycin ml . Mutagenesis and plasmid construction. Mutagenic PCRs were performed on a plasmid carrying a 232 bp Spe I/ Sna BI fragment, containing the petH 5′-non-coding region from Synechocystis , using mutagenic primer pairs 5′-GGTAAATCTAGACATGGGTTAC3′/5′-GTAACCCATGTCTAGATTTACC-3′ for the X mutation and 5′-CGGTTATAAGATACATGGGTTAC-3′/5′-GTAACCCATGTATCTTATAACCG-3′ for the P mutation. The resulting plasmids were sequenced to verify the fidelity of the PCR amplification. To create the cargo plasmids, the modified Spe I/ Sna BI fragments were used to replace the corresponding WT fragment in pBX, a plasmid carrying a 900 bp fragment of petH with the Ω cassette inserted 70 bp upstream from the putative NtcA-binding site – an insertion shown to be neutral to petH expression (Omairi-Nasser et al. , 2011). Cargo plasmids were introduced into WT by genetic transformation and antibiotics were used for the selection of the desired mutation. Complete segregation of the mutant alleles was confirmed by PCR, and in some cases the PCR products were subjected to restriction analysis and sequencing to ascertain the identity of the amplified fragments. Recombinant NtcA expression, purification and gel retardation assays. An Nde I/ Xho I fragment encompassing the entire Synechocystis ntcA gene was synthesized by PCR from genomic DNA using the oligonucleotides 5′-CTAGCATATGGATCAGTCCCTAACCC3′/5′-GCTACTCGAGGGTAAACTGTTGACTGAG-3′. This fragment was cloned into the pET24a(+) plasmid (Novagen) to generate pSNtcA. Exponentially growing Escherichia coli BL21 cells transformed with pSNtcA were treated with 0.5 mM IPTG for 4 h. The C-terminal His-tagged version of Synechocystis NtcA was purified by nickel-affinity chromatography using His-Bind matrix (Novagen) following the manufacturer's instructions. For further purification, the sample was subjected to gel filtration chromatography using a HiLoad 16/60 Superdex 75 column (GE Healthcare) running on an Akta FPLC system. Synechocystis NtcA–His6, expressed and purified as described above, was used in gel retardation assays. The P petH promoter probes were obtained by Spe I/ Sna BI digestion of plasmids carrying WT or modified versions of the NtcA-binding site. DNA fragments were end-labelled with [α-P]dCTP using Sequenase version 2.0 enzyme. The binding reactions and electrophoresis were carried out as described previously (Muro-Pastor et al. , 1996). Electrophoresis and immunoblotting of cell extracts. The experiments were performed as in our previous work (Omairi-Nasser et al. , 2011). For Synechococcus PCC7002 and Anabaena PCC7120 FNR −2−1 −1 −1−1 −1 −1 32 View larger version: In this page In a new window Download as PowerPoint Slide View larger version: In this page In a new window Download as PowerPoint Slide immunodetection, the amount of total-cell extracts loaded per well was doubled compared with Synechocystis (evaluated by chlorophyll concentration to 1 µg) and the antibody (raised against the Synechocystis FNR) was two times more concentrated (1/5000) than it was for Synechocystis (1/10 000). Anabaena heterocysts were separated from vegetative cells according to Golden et al. (1985). Results NtcA binding to WT and modified versions of the Synechocystis petH promoter To test whether NtcA binds to the Synechocystis petH promoter, electrophoretic mobility shift assays (EMSAs) using purified Synechocystis NtcA protein were performed. The protein was expressed in E. coli and purified as a His-tagged version. Binding assays were performed using an Spe I/ Sna BI DNA fragment, which spans positions −85 to +144 with respect to the distal petH transcription start point. It has been claimed that >8 nt might separate the NtcA-binding triplets (Jiang et al. , 2000), so two putative binding sites (GATN8TAC and GTGN10TAC), centred at −41.5 and −42.5 upstream from the distal petH transcription start point, were considered. We tested fragments containing modified versions of both putative sites; X, creating an Xba I restriction site, and P, creating a Psi I site where only the putative site N10 was modified (Fig. 1). FF i i g g . . 1 1 . . NtcA binds to the Synechocystis petH promoter in vitro . EMSAs were performed with a 232 bp DNA fragment, encompassing the distal and the proximal petH promoters. The (a) WT fragment or mutant fragments, (b) X and (c) P, carrying changes to the grey bases, were used for binding reactions. Sequences of the putative NtcA-binding site, with the consensus triplets underlined when present, are shown below each EMSA. DNA probes (0.86 nM) were incubated in the presence of purified NtcA protein (0.1–3.0 µM). When purified NtcA was incubated with the WT-labelled probe, an NtcA–DNA complex was clearly detected (Fig. 1a); however, when the X probe was used, no NtcA–DNA complex was detected (Fig. 1b). The mutations present in the P probe did not prevent NtcA binding in vitro (Fig. 1c). These results indicate that NtcA binds in vitro to the GATN8TAC site, which is centred at −41.5 upstream from the distal petH transcription start point. NtcA binding is required for FNRS accumulation in Synechocystis To determine whether the identified NtcA-binding site operated in vivo , the X mutation linked to the Ω cassette was introduced in Synechocystis , yielding mutant NBX (Fig. 2a). Total segregation of the chromosomes was confirmed by PCR and restriction analysis, in which the presence of the Xba I site confirmed the integration of the modified NtcA-binding site into the segregated chromosome (Fig. 2b). FF i i g g . . 2 2 . . Construction and characterization of a mutant lacking the NtcA-binding site. (a) Structure of the mutagenized petH gene; a solid line represents the 5′-non-coding region where the X mutation, which abolished NtcA binding (two black boxes) and created an Xba I site ( Xba I*), is linked to the Ω cassette. A black arrow represents the petH ORF and half arrows indicate the location of PCR primers. (b) PCR and restriction analysis of the mutant: –, PCR produced a 1 kb fragment from the WT and 3 kb fragments from BX and NBX, due to the Ω cassette; +, Xba I digestion of the PCR products yielded an additional 0.4 kb fragment in NBX. (c) Immunodetection of the FNR in cell extracts from the WT, BX and View larger version: In this page In a new window Download as PowerPoint Slide View larger version: In this page In a new window Download as PowerPoint Slide NBX under standard conditions (0), and upon nitrogen starvation for 24 and 72 h. Unlike WT and BX, NBX failed to induce FNRS synthesis. FNR isoform accumulation upon nitrogen starvation was examined in totalprotein extracts from NBX, which carried the X mutation plus an Ω cassette insertion 115 bases upstream from the distal transcription start point, the WT and BX, a strain carrying a WT petH allele plus the omega cassette at the same site as NBX. Fig. 2(c) shows that, unlike the WT and BX, NBX did not accumulate FNRS even after 3 days of nitrogen starvation. A similar experiment performed with the P mutation showed a WT behaviour for FNR isoform accumulation, which excludes NtcA binding to the putative N10 site in vivo (data not shown). We have shown previously that FNRS accumulation depends on the petH distal promoter, producing a long transcript whose translation yields FNRS (OmairiNasser et al. , 2011). The absence of FNRS in a strain lacking the NtcA-binding site implies that NtcA is required for petH transcription from the distal promoter. These results confirmed the requirement of the longer transcript for the synthesis of FNRS and established its transcription activation by NtcA. FNRS accumulation in Synechococcus sp. PCC7002 Recent transcriptome studies in Synechococcus sp. PCC7002 showed that petH transcription proceeds from a proximal transcription start point, located 87 bases upstream from the ORF, under standard conditions, and from a distal transcription start point, located 283 bases upstream from the ORF, operating under nitrogen starvation (Ludwig & Bryant, 2012). We performed Western blots on total extracts from Synechococcus sp. PCC7002 grown under standard and nitrogen starvation conditions. Fig. 3 shows that FNRS was expressed upon nitrogen starvation, consistent with the transcript results. FF i i g g . . 3 3 . . FNR immunodetection in cell extracts from WT Synechococcus sp. PCC7002 grown with nitrate (0) or nitrogen starved for 96 h (two amounts were loaded from each sample); the last well contains extracts from WT Synechocystis starved for 24 h for comparison. A putative NtcA-binding site was found upstream from the distal petH promoter in Synechococcus sp. PCC7002 (Fig. 6 a, b ). Thus, the situation in this cyanobacterium turns out to be similar to the situation described for Synechocystis , where FNRS accumulation follows an NtcA-induced and longer petH transcript. FNRS accumulation in Anabaena PCC7120 In Anabaena PCC7120, petH is transcribed from a constitutive transcription start point, located 63 bases upstream from the ORF, and from an NtcA-regulated transcription start point, located 188 bases upstream from the ORF (Valladares et al. , 1999). To test whether the NtcA-regulated long transcript is translated into a small FNR isoform, we performed Western blots on total extracts from the WT as well as from an ntcA insertional mutant (CSE2; Frías et al. , 1994) grown with ammonia or in the absence of combined nitrogen. Fig. 4 shows that FNRS accumulates upon nitrogen step-down in WT cells, whilst it failed to accumulate in the ntcA mutant. A small amount of FNRS was detected in the presence of nitrogen in the WT (7120WT, 0), but also when NtcA was absent (NtcA , all wells). FF i i g g . . 4 4 . . FNRS accumulation in Anabaena sp. PCC7120. FNR immunodetection in WT and NtcA-deficient mutant, CSE2, grown with ammonium (0) or in the absence of combined nitrogen for 8 and 24 h. As the distal promoter was shown to be the main promoter operative in the heterocysts (Valladares et al. , 1999), purified heterocysts were tested for the presence of FNRS. Fig. 5(b) shows clearly that FNRS is the major isoform in purified heterocysts. Heterocyst extract purity was estimated by evaluating − View larger version: In this page In a new window Download as PowerPoint Slide their relative content of ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit (RbcL, which is expressed only in vegetative cells). As shown in Fig. 5, isolated heterocysts had similar amounts of RbcL and FNR L, which suggested that mature heterocysts contain almost exclusively FNRS. FF i i g g . . 5 5 . . Anabaena sp. PCC7120 expresses FNRL in vegetative cells and FNRS in heterocysts. Different amounts of total extracts were migrated using SDS-PAGE. The first two lanes were Coomassie stained (a), and the rest were blotted on a membrane and hybridized with an FNR antibody (b); the membrane was striped and rehybridized with an RbcL antibody to evaluate heterocyst purity (c). Numbers to the right of the Coomassie-stained gel and to the left of the blots indicate the sizes of the molecular mass markers; R, P and N indicate possible positions of specific and abundant proteins (RbcL, phycocyanin and nitrogenase, respectively). Discussion This work shows that petH regulation is similar in cyanobacteria capable of FNRS synthesis. An ORF-proximal transcription start point produces a transcript that is translated into FNRL, whilst a distal transcription start point controlled by NtcA, produces a longer transcript that is translated into FNRS. When Synechocystis is grown under standard conditions, FNRL is the major isoform but traces of FNRS are present. The distal petH transcription start point was detected in addition to the proximal transcription start point under standard conditions in the genome map of Synechocystis transcription start points (Mitschke et al. , 2011a). Here, we show that NtcA binding to the distal promoter is required for FNRS synthesis that depends on the distal petH promoter activity (Omairi-Nasser et al. , 2011). NtcA was shown to be essential for Synechocystis under all known conditions (García-Domínguez et al. , 2000), which implies that NtcA is present even when nitrogen is available. Therefore, the slight accumulation of FNRS and the presence of the longer transcript in the WT Synechocystis are due to the presence of NtcA under standard conditions. A slight difference was found in Synechococcus sp. PCC7002. Whilst FNR S accumulates under nitrogen starvation, it was not detected under standard conditions. In a recent transcriptome work, the long petH transcript, in addition to the short transcript, was detected under standard conditions (Ludwig & Bryant, 2012; M. Ludwig & D. A. Bryant, personal communication). The absence of FNRS, under the standard conditions used in our work, might either result from a poor recognition of the Synechococcus sp. PCC7002 FNR S, by the Synechocystis antibody, or from the different growth conditions used in each laboratory. The low level of FNRS detected in Anabaena grown in the presence of ammonia, as well as in the NtcA-deficient mutant (Fig. 4), suggests that NtcA does not tightly control transcription from the distal promoter in this cyanobacterium. It is noteworthy that a putative −35 element overlaps the NtcA-binding site in Anabaena (Fig. 6a, c ); this might be responsible for the low level of FNRS detected in the absence of NtcA. It was shown that NtcA was responsible for the upregulation of the distal petH promoter in Anabaena (Valladares et al. , 1999); here, we showed that NtcA is required for the accumulation of a higher level of FNRS. FF i i g g . . 6 6 . . Structure of the NtcA-binding sites associated with the distal petH promoters. (a) The consensus NtcA-binding site is shown in bold upper-case letters. NtcA-binding sites found at the distal petH promoter in a Synechocystis sp. PCC6803, b Synechococcus sp. PCC7002 and c Anabaena sp. PCC7120. Bold upper-case letters indicate nucleotides that are identical to consensus sequence; lower-case letters indicate nucleotides that might interact with the conserved Arg192 of NtcA. An arrowhead indicates the symmetry axis of the NtcA-binding site, which is located −41.5, −40.5 and −42.5 upstream from the distal petH transcription start point (TSP) in a , b and c, respectively. The underlined sequence in c could constitute a −35 promoter element in Anabaena sp. PCC7120; View larger version: In this page In a new window Download as PowerPoint Slide boxed sequences represent putative −10 promoter elements. (b) Summary of the proposed NtcA F-helix residue interaction with the site. NtcA arginine residues (the numbers are those of Synechocystis ) are labelled R and R′ to indicate that they belong to different subunits of the dimer. In addition to its photoautotrophic growth ability, Anabaena can fix molecular nitrogen when combined nitrogen is absent. Differentiated cells called heterocysts achieve this function for the adjacent vegetative (photosynthetic) cells, which in turn supply carbohydrates to the non-photosynthetic heterocysts. Heterocysts have been shown to contain 14 times more FNR than vegetative cells (Razquin et al. , 1996). Since ~10 % of the cells in a nitrogenfixing culture are heterocysts, the equal amounts of FNRS and FNR L in a nitrogen-fixing culture (Fig. 4, 7120WT, 24) indicate that FNRS is associated with heterocysts. In another experiment, we showed that purified heterocysts contained almost exclusively FNRS, whilst FNR L is the major isoform in ammonia-grown vegetative cells. This suggests that FNRL function is related to photosynthesis and sugar anabolism, whilst FNRS functions in an environment where sugar catabolism is activated to sustain nitrogen fixation. Obligate photoautotrophs like Synechococcus elongatus and Thermosynechococcus elongatus do not contain a second initiating codon and do not accumulate FNRS (Thomas et al. , 2006). In a recent transcriptome study of S. elongatus , the petH transcription start point was located 135 bases upstream from the ORF under standard conditions (Vijayan et al. , 2011). Unfortunately, no transcription data are available for this cyanobacterium under nitrogen-limited conditions. However, the fact that strains capable of FNRS synthesis such as Synechocystis sp. PCC6803, Synechococcus sp. PCC7002 and Anabaena sp. PCC7120 are facultative heterotrophs strongly suggests that FNRS function is associated with catabolism. Potential interaction of NtcA with its binding site Comparison of the petH NtcA-binding sites reveals a possible role for the first nucleotide position after the consensus triplet. As in the Crp structure, the F-helix of NtcA ensures DNA recognition. The F-helix of Crp was shown to exhibit sequence preferences at specific positions (GTGA) within each DNA half-site, which are mediated by Arg180, Glu181 and Arg185 (Lawson et al. , 2004). In the Crp–DNA complex, Arg180 interacts with guanine ( O and N atoms) of the GC pair at position 1 of its consensus; a similar interaction may occur in the NtcA–DNA complex through Arg187 (or 186, depending on amino acid numbering) (position 1, Fig. 6b). In place of Glu181 of Crp, a Val is found in NtcA, which is believed to accommodate an AT (instead of a GC) pair at position 3 and explains the GTA triplet in the NtcA-binding consensus (Llácer et al. , 2010). However, Arg185 in the Crp–DNA complex interacts with guanine ( O and/or N ) of GC at position 3, but also with thymine ( O ) of the AT pair at position 4 (Lawson et al. , 2004). In the NtcA complex, a similar interaction could occur between Arg192 (or 191) and the adenine ( N ) of the AT pair at position 3 (Fig. 6b). In this context, NtcA binding would be strengthened by the presence of an AT pair at position 4, as Arg192 would also interact with the thymidine ( O ). In Synechocystis , the position 3 AT pair is not found in the 5′ half of the petH NtcA-binding site, but an AT pair is present in both half-sites at position 4 (Fig. 6a, a ). In Synechococcus PCC7002, the position 3 AT pair is not conserved in the 3′ half, but again both half-sites retained an AT pair at position 4 (Fig. 6a, b ). In Anabaena , none of the position-3 AT pairs are conserved and only the one at position 4 of the 3′ half is conserved (Fig. 6a, c ). Therefore, the first nucleotide after the GTA consensus triplet could play a role in NtcA binding. Further experiments are required to test this proposal. Acknowledgements We are indebted to Alicia M. Muro-Pastor for providing the Western blot used in Fig. 4. The work in France was financially supported by the Centre National de la Recherche Scientifique (UMR8221) and the Commisariat à l'Energie Atomique (Bioénergies DSV, IBITEC-S). The work in Spain was supported by Ministerio de Economía y Competitividad co-financed by FEDER (grant BFU2010-15708) and 6 7 6 7 4 7 4 by Junta de Andalucía (grant P07-CVI-02792 and group BIO-284). The anonymous reviewers are acknowledged for their constructive comments. BFU2010-15708FEDER Centre National de la Recherche Scientifique Commisariat à l'Energie Atomique Ministerio de Economía y Competitividad Junta de Andalucía References Frías J. E., Flores E., Herrero A. ((11 99 99 44 )).. 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