A second isoform of the ferredoxin:NADP oxidoreductase generated by an in-frame initiation of translation

Abstract

Ferredoxin:NADP oxidoreductases (FNRs) constitute a family of flavoenzymes that catalyze the exchange of reducing equivalents between one-electron carriers and the two-electron-carrying NADP(H). The main role of FNRs in cyanobacteria and leaf plastids is to provide the NADPH for photoautotrophic metabolism. In root plastids, a distinct FNR isoform is found that has been postulated to function in the opposite direction, providing electrons for nitrogen assimilation at the expense of NADPH generated by heterotrophic metabolism. A multiple gene family encodes FNR isoenzymes in plants, whereas there is only one FNR gene (petH) in cyanobacteria. Nevertheless, we detected two FNR isoforms in the cyanobacterium Synechocystis sp. strain PCC6803. One of them (FNR_S ≈34 kDa) is similar in size to the plastid FNR and specifically accumulates under heterotrophic conditions, whereas the other one (FNR_L ≈46 kDa) contains an extra N-terminal domain that allows its association with the phycobilisome. Site-directed mutants allowed us to conclude that the smaller isoform, FNR_S, is produced from an internal ribosome entry site within the petH ORF. Thus we have uncovered a mechanism by which two isoforms are produced from a single gene, which is, to our knowledge, novel in photosynthetic bacteria. Our results strongly suggest that FNR_L is an NADP⁺ reductase, whereas FNR_S is an NADPH oxidase.

Cyanobacteria and chloroplasts, the eukaryotic organelles derived from cyanobacteria, are defined by their ability to carry out the oxygenic photosynthesis required for their photoautotrophic growth. Although some cyanobacterial strains can grow chemoheterotrophically in the dark at the expense of external carbohydrates, their chemoheterotrophy is usually restricted to the mobilization of reserves during dark periods or to specialized cells like heterocysts.

Ferredoxin:NADP oxidoreductases (FNRs) catalyze the exchange of reducing equivalents between ferredoxin (or flavodoxin) and NADP(H). In cyanobacteria and photosynthetic plastids (chloroplasts) the main role of the FNR is to catalyze the final step of photosynthetic electron transport, providing NADPH for CO₂ assimilation and other reductive metabolism. In nonphotosynthetic root plastids a genetically distinct FNR isoform is postulated to function in the opposite direction, providing electrons for nitrogen assimilation at the expense of NADPH generated by heterotrophic metabolism (1, 2). It has been suggested, for both cyanobacteria and plastids, that FNR could participate in respiration and cyclic electron transfer as an NADPH dehydrogenase (3–6).

Despite the similarity in both biochemical properties and protein structure for the cyanobacterial and plastid FNR (7), the unique petH gene in most phycobilisome (PBS)-containing cyanobacteria, except Gloeobacter violaceus (8) and Synechococcus spp. strains OS-A and OS-B′ (9), encodes a ≈46-kDa FNR that contains an N-terminal domain, of ≈80 aa, whose sequence is similar to PBS rod-linker polypeptides (10). The PBS is a large and abundant bilin–protein complex that harvests light for photosynthesis. Nondiazotrophic cyanobacteria respond to the lack of combined nitrogen by slowing down photosynthesis and anabolic reactions, while catabolizing reserves to survive prolonged periods of starvation (11). During nitrogen starvation, PBS degradation supplies amino acids for the synthesis of essential proteins, while also reducing light harvesting for photosynthesis (12).

Purified PBS from several cyanobacterial strains contain significant amounts of the 46-kDa FNR, one to two molecules per PBS (10, 13, 14). It was proposed that the FNR binds to the PBS rods via its linker domain to fulfill functions in both cyclic electron transport and respiration, in close proximity to the membrane (6, 15). A ≈35-kDa FNR (similar in size to that of plastids) has been purified from several cyanobacterial strains, Spirulina sp. (16), Anabaena cylindrica (17), and Synechocystis sp. strain PCC6803 (18). This result was attributed to proteolytic degradation of the N-terminal domain of the 46-kDa FNR (6, 10).

In this article, the use of a highly specific antibody together with genetic and physiological studies reveals that two FNR isoforms are produced by differential translation initiation of the petH ORF in Synechocystis sp. PCC6803 (hereafter called Synechocystis). One of these isoforms accumulates under conditions of heterotrophic metabolism. This phenomenon is shown to occur only in cyanobacteria capable of heterotrophy.

Results

FNR Undergoes Proteolysis in Rodless PBS Mutants.

In an attempt to clarify the FNR localization issue we examined phycobiliprotein (PBP) complexes purified from PBS-deficient mutants: CB (ΔcpcC1C2), in which the lack of two rod linkers (L_R³³ and L_R³⁰) results in PBS containing only one phycocyanin (PC) hexamer per rod instead of three in the WT (19), CK (ΔcpcBAC1C2) where the PBS is restricted to the allophycocyanin (APC)-containing core (B.U. and G.A., unpublished work), and AB (ΔapcAB) producing PC only and assembling uncoupled rods (20) (Fig. 1A). PBS from CB and WT contained similar amounts of FNR, whereas none was visible in PBS from CK. Rods purified from AB contained a major contamination in the 45- to 50-kDa range that prevented observation of the FNR (Fig. 1B). Immunological analysis with an FNR-specific antibody confirmed the absence of FNR in purified cores from CK and revealed its presence in rods from AB (Fig. 1B′). We probed total cell extracts from the WT and four PBS-mutants: CB, CK, AB, and PAL (PC⁻, ΔapcAB, ΔapcE), a mutant totally devoid of PBPs (21). In WT, CB, and AB extracts two isoforms were detected at 46 and 34 kDa, which we designated FNR_L and FNR_S, respectively (Fig. 1C′). In CK and PAL cell extracts FNR_L seemed to undergo proteolysis (Fig. 1C′) as has been observed (10). It should be noted that cell extracts from AB, PAL, and CK contain relatively high amounts of FNR_S, a point that is addressed in Discussion. The fragments detected in extracts from rodless mutants were reminiscent of the profile of the Anabaena sp. FNR produced in Escherichia coli (22); by the use of a proteinase-deficient mutant, Martinez-Julvez et al. (22) eliminated the intermediary products and obtained two polypeptides of 49 and 36 kDa. Those results and our results suggest that FNR_L undergoes proteolysis when not bound to the PBS, whereas FNR_S appears not be a proteolytic product of FNR_L.

Fig. 1.

Characterization of the FNR in purified PBP complexes and total extracts from Synechocystis. (A) Representation of PBSs in the WT, a mutant bearing one PC hexamer per rod (CB), a PC-deficient mutant (CK), and PC-hexamer structures in the APC-deficient mutant (AB). PC hexamers are represented as dark gray cylinders and APC-containing cores are represented as three light-gray circles. (B) Polypeptide composition of purified PBP complexes. (B′) Western blot analysis of the FNR in the same PBS samples separated on an identical gel loaded with four times less sample per well. (C) Coomassie-stained PAGE of total cell extracts from the above strains plus extracts from PAL, totally devoid of PBP. (C′) Western blot analysis of the FNR in the same extracts separated on an identical gel loaded with half as much sample per well. The identities of the PBS subunits are labeled on the left and approximate masses are labeled in kDa in the middle. L_X, linker polypeptide located at position X, where X can be R (rod), RC (rod core), C (core), or CM (core membrane); linkers with identical location are distinguished by a superscript indicating their mass.

FNR_S Accumulates When Photosynthesis Is Slowed Down.

To determine the origin of FNR_S in Synechocystis, we tested the effect of nitrogen starvation (known to induce PBS degradation) on the FNR in cell extracts from WT and two mutants in which PBS were not degraded under nitrogen starvation conditions: N1 lacking nblA1, a gene that is induced and necessary for PBS trimming under nitrogen starvation (23), and M55 deficient in ndhB, encoding a subunit of the NDH-I complex (24). The latter mutant exhibits attenuated PBS degradation when nitrogen-starved, probably because of deficient respiration (J.-C.T. and G.A., unpublished work). Under the nitrogen-starvation conditions used in our experiment, leading to the loss of two rod-linkers, L_R³³ and L_R³⁰, and their associated PC in the WT, FNR_L did not undergo proteolysis in the WT nor in either nonbleaching mutant. FNR_S accumulates in all three strains, although to a slightly lesser extent in the mutants (Fig. 2). We also probed total extracts from the WT grown under different growth environments: photoheterotrophic (light, 3-(3,4-dichlorophenyl)-1-1 dimethylurea, glucose), chemoheterotrophic (dark, glucose), mixotrophic (dim-light, glucose), and iron starvation. Again, although no intermediary proteolysis of FNR_L was observed, the relative amount of FNR_S increased significantly in cells grown under heterotrophic and iron-starvation conditions compared with cells grown under photoautotrophic conditions (Fig. 3).

Fig. 2.

Characterization of the FNR during nitrogen starvation (for 0, 16, and 34 h) in two mutants unable to degrade PBS, N1 and M55, compared with the WT. (A) Coomassie-stained PAGE of total extracts. (B) Western blot analysis of the FNR in the same extracts separated on an identical gel loaded with half as much sample per well.

Fig. 3.

Immunodetection of the FNR in cell extracts of Synechocystis grown under: PA, photoautotrophic; PH, photoheterotrophic; CH, chemoheterotrophic; MX, mixotrophic; −Fe, iron starvation conditions.

FNR_S Derives from a Second Translation Initiation Site.

The unique petH gene in Synechocystis encodes a polypeptide of 413 aa (46 kDa) and was shown to be transcribed into a single mRNA (25), indicating that FNR_S is generated posttranscriptionally. A putative translation start is found at Met-113, which would produce an FNR_S with the right size (34 kDa) and an N-terminal sequence matching the one found by Matsuo et al. (18) for an FNR that copurifies with the NDH-1 complex in Synechocystis. Although this may sound curious, many examples of internal ribosome entry sites (IRESs) within coding regions have been described in various organisms, although rarely in prokaryotes (26–28). To test this hypothesis, we introduced missense and frame-shift mutations in petH by a procedure similar to that described in ref. 6. Each construct (described in Methods and Fig. 8, which is published as supporting information on the PNAS web site) was used to transform WT Synechocystis where double recombination led to its integration into the chromosome. The fully segregated mutant carried the modified petH gene and an antibiotic-resistance cassette. The missense mutation in MI6 changed the putative start methionine into an isoleucine. Frame-shift mutations, created by single base deletion or insertion, caused premature translation stops upstream and downstream of Met-113 in FS1 and FS2, respectively (Fig. 4A). PCR, restriction analysis, and DNA sequencing confirmed the genotypes of the mutants (data not shown).

Fig. 4.

FNR_S is translated from Met-113 in Synechocystis. (A) (Upper) Schematic representation of the FNR_L polypeptide with the linker, hinge (H), and enzymatic (FNR) domains. Start Met-1 and Met-113 are shown. Met-113 was changed to isoleucine (ATC) in MI6. Frameshifts in FS1 and FS2 result in translation termination (∗). (Lower) Translated polypeptides are indicated. (B) Gel electrophoresis and immunoblot of total protein extracts and purified PBS. In the WT FNR_L and FNR_S (unusually abundant in this sample) are detected, whereas only FNR_L is present in MI6, the week secondary band is the product of either proteolysis or functionally insignificant translation initiation at codon 102; GTG). Only FNR_S is present in FS1 and FS2 (the week secondary bands are probably aggregation products of FNR_S; they are absent in the FS1 extracts presented in Fig. 5). Comparison of PBS purified from WT to those from FS1 confirms the absence of FNR_L in FS1.

As shown in Fig. 4B, cell extracts from mutant MI6 contained only FNR_L, consistent with FNR_S originating from either an IRES at Met-113 or from a specific cleavage of FNR_L just upstream of this residue. In cell extracts from FS1 and FS2 (where translation initiated at Met-1 terminates before or just after Met-113, respectively) FNR_S was present at the WT FNR_L level, whereas FNR_L was absent (Fig. 4B, FS1 and FS2). Therefore, FNR_S must be produced from Met-113 and not by proteolysis. Furthermore, comparison of PBS purified from FS1 to those from the WT clearly shows that the FNR was no longer associated with PBS in the absence of the rod-linker domain (Fig. 4B, PBS), in agreement with previous experiments in which 75 aa were deleted from FNR_L (14).

We tested the effect of nitrogen starvation on the FNR in these mutants. Fig. 5 clearly shows that FNR_S did not accumulate in MI6 under nitrogen starvation, although clear PBS trimming occurred. FS1 contains only FNR_S and its levels seem to increase during nitrogen starvation. FS1 cultures appeared to bleach faster than the WT and MI6.

Fig. 5.

Impact of nitrogen starvation (for 0, 6, and 34 h) on the FNR in total extracts from the WT and mutants producing only FNR_L (MI6) or FNR_S (FS1). (A) Coomassie-stained PAGE of total extracts. (B) Western blot analysis of the FNR in the same extracts separated on an identical gel loaded with half as much sample per well.

Compared with the WT, no difference in growth rate was observed when MI6 was grown under photoautotrophic conditions whereas FS1 growth was significantly slowed down (25% longer doubling time). Under photoheterotrophic conditions MI6 growth was slowed down whereas FS1 growth was similar to that of the WT.

FNR_S Is Absent in Cyanobacteria Lacking a Second Methionine.

FNR contains a PBS-linker domain in all PBS-containing cyanobacterial strains from which petH has been sequenced, except for G. violaceus (8) and Synechococcus spp. strains OS-A and OS-B′ (9). We therefore wondered whether a second translation initiation site was associated with the presence of a linker domain in the FNR. Alignment of FNR sequences from different strains indicates that a putative initiating Met is present in Anabaena sp. strain PCC7120 and Synechococcus sp. strain PCC7002, whereas it is absent in the obligate photoautotrophes: Synechococcus elongatus and Thermosynechococccus elongatus (Fig. 6). The absence of a putative initiating site is also observed in all marine cyanobacteria of the genus Synechococcus sequenced to date (F. Partensky, personal communication), whereas it is present in petH of the marine diazotroph Trichodesmium erythraeum (Joint Genome Institute gene ID 4667). We probed cell extracts from strains lacking a putative second initiating site (T. elongatus and S. elongatus) and from G. violaceus as a control. Fig. 7 shows that no trace of a second FNR signal is detected in any of these strains even under nitrogen-starvation conditions. As expected G. violaceus contained only FNR_S. Cell extracts from Anabaena PCC7120 and Synechococcus PCC7002 exhibit, as in Synechocystis, two FNR isoforms (data not shown).

Fig. 6.

Sequence alignments of six cyanobacterial FNR (N-terminal part) exhibits the three domains (rod-linker, hinge, and catalytic). Strains: ANA, Anabaena PCC7120 (P58558); SP6, Synechococcus elongatus (Q5N4L3); TEL, Thermosynechococccus elongatus BP-1 (Q93RE3); S70, Synechococcus PCC7002 (P31973); SY3, Synechocystis PCC6803 (Q55318); and GVI, G. violaceus (Q7NI88). Amino acids are shown in boldface at positions where they are identical in >50% of the sequences compared (by Clustal W). The first initiating Met is italicized and the second one is underlined when present. A PEST-like sequence (highlighted) is found only in Synechocystis.

Fig. 7.

Cyanobacteria lacking the second putative initiating methionine, T. elongatus (T) and S. elongatus (S), contain only FNR_L even after 38 h of nitrogen starvation (−). G. violaceus contains only FNR_S (G).

Discussion

By the use of site-directed mutants in Synechocystis, we demonstrate that two FNR isoforms are produced by differential initiation of translation. That mutant MI6 produced only FNR_L whereas mutants FS1 and FS2 produced only FNR_S argues against the widely held view that FNR_S is a product of proteolytic cleavage of FNR_L and demonstrates that the small isoform is the result of a second translation initiation, at 337-nt distance from the first. To our knowledge this demonstration of IRES is unique in photosynthetic prokaryotes. Another bacterial example is found in E. coli where cheA (third locus in the mocha operon) encodes two proteins (CheA_L and CheA_S) translated from two in-frame start sites, the functional significance of these isoforms and the translation-initiation mechanism involved are not clear (29).

In Anabaena spp. petH produces two mRNAs; one is constitutive (starts at nucleotide −63 from Met-1) and the other one is used in the absence of combined nitrogen (starts at nucleotide −188 from Met-1) (30). This larger transcript is expected to produce FNR_S by a yet-unknown posttranscriptional mechanism that would prevent initiation at Met-1. The organization reported for petH in Synechocystis is markedly different because van Thor et al. (25) mapped only one transcription-start point at nucleotide −523 from Met-1. Although the nucleotide sequences incorporating the initiation codon are known to determine translation initiation efficiency in prokaryotes (31), initiation cannot be evaluated from knowledge of the sequence alone. An SD sequence is involved in translation initiation but might not be required when the initiation codon is located within an AT-rich sequence that forms no stable secondary structure (32), which is the case for the sequence incorporating Met-113 in the Synechocystis petH. It is feasible for proteins or other sRNA to bind to an mRNA and alter its translation efficiency. Alternatively, a low level of translation can also allow RNA secondary structures to form, causing premature termination, frameshifting, and initiation of transcription at codons that are not used when translational level is high (33). The inverted repeats present at the 5′ end of petH mRNA in Synechocystis suggest that secondary structures are likely to form; mutations within these repeats could clarify the issue.

Proteolysis occurs in the linker domain of FNR_L in the absence of PC, just as PBS linkers are known to be highly sensitive to proteolysis when not associated to PBPs. This phenomenon occurs in Synechocystis mutants lacking PC and during heterologous expression of FNR_L in E. coli (22); we found similar proteolytic fragments in extracts from PC-deficient mutants of Synechocystis and E. coli expressing petH (Fig. 9, which is published as supporting information on the PNAS web site), which indicates the action of similar proteases in both bacteria. The sizes of the FNR_L proteolytic fragments also suggest that proteolysis occurs within the linker domain of the FNR and not only at a putative PEST site as has been proposed (6, 10). Furthermore, heterologous expression of the mutant alleles MI6 and FS failed to produce FNR_S and FNR_L, respectively, indicating that also in E. coli translation initiates at both sites (Fig. 9). We also constructed a PC-deficient mutant of Synechocystis carrying the MI6 mutation; in this strain FNR_S is absent and FNR_L exhibits the proteolytic profile characteristic of the absence of PC (Fig. 10, which is published as supporting information on the PNAS web site).

FNR_L might have been created in an ancestral cyanobacterium (like G. violaceus) by fusion of genes encoding PBS linker and FNR_S. We show here that the second initiating methionine is not required for photosynthesis, which might explain its loss in cyanobacteria that are obligate phototrophs. Its presence in facultative heterotrophs suggests a separate function for the small isoform in these organisms. We also show that different physiological conditions alter the relative amounts of the two FNR isoforms, which has implications for their possible functions. FNR_L seems clearly related to photosynthetic electron transfer, while FNR_S accumulates under heterotrophic and starvation conditions where catabolism is stimulated and anabolism slowed down.

Plastidic FNR seem to have adapted their catalytic efficiency to the host tissue; leaf FNR satisfy the requirement of electron flow to sustain CO₂ fixation, while root FNR acts as a shuttle between the NADPH generated by heterotrophic metabolism and electron-accepting enzymes, i.e., nitrite reductase (34). FNR_L attachment to the PBS, in cyanobacteria, might provide a way for the FNR to optimize electron flow for CO₂ fixation (NADPH production); the slow photoautotrophic growth of mutants lacking FNR_L (FS) supports this hypothesis. FNR_S accumulation, in the WT during heterotrophic growth, and the impairment of heterotrophic growth in MI6, suggest that the attachment to PBS is a hindrance for the FNR activity required under these conditions (NADPH consumption). It is possible that in mutants such as CK, AB, and PAL, containing a higher photosystem II/photosystem I ratio caused by PBS deficiencies (20, 21), FNR_S accumulation (visible in Fig. 1C′) is caused by an excess of NADPH. This hypothesis is strengthened by the accumulation of FNR_S in the WT grown under high light (Fig. 11, which is published as supporting information on the PNAS web site). These results strongly suggest that FNR_L is an NADP⁺ reductase, whereas FNR_S is an NADPH oxidase.

The fact that the obligately phototrophic cyanobacteria tested here express only FNR_L and that FNR_S accumulates in Synechocystis (and most probably other facultatively heterotrophic cyanobacteria) under conditions where heterotrophic metabolism is needed raises the old issue of FNR being the dehydrogenase part of the NDH-I complex in cyanobacteria and plant plastids (3, 5, 18).

Methods

Strains and Growth Conditions.

WT and mutants of Synechocystis were grown as in ref. 19. For nitrogen starvation, cells were harvested by centrifugation and resuspended in a medium where NaCl replaced NaNO₃.

Preparation of Cell Extracts.

Cells were centrifuged, washed with 50 mM EDTA, and resuspended in 20 mM Tricine, pH 8 containing Complete protease inhibitor (Roche, Meylan, France), then broken by vortexing 6 min with glass beads. Unbroken cells and glass beads were removed by centrifugation at 7,500 × g for 2 min. The supernatant was used as total cell extract. PBSs were purified as in ref. 35.

Gel Electrophoresis and Western Blotting.

Proteins were separated by using Tris-Tricine lithium dodecyl sulfate-12% PAGE. Proteins were trichloroacetic acid-precipitated, and the pellet was resuspended in the loading buffer. Chlorophyll concentration was used to ensure equivalent loading of cell extracts, 2 or 4 μg chl was loaded per 6-mm well for blotting or Coomassie staining, respectively. For immunoblots, proteins were transferred to PVDF membranes by using semidry transfer. Blots were blocked with Tris-buffered saline supplemented with 0.1% Tween and 0.5% dry skimmed milk and incubated with the primary antibody (1:5,000 dilution). After washing, blots were incubated 2 h at room temperature with a 1:15,000 dilution of peroxidase-conjugated anti-rabbit IgG (Promega, Madison, WI). The signal was visualized by using ECL chemiluminescent substrate (Amersham, Piscataway, NJ) and autoradiography films. Images were generated by using a CCD camera and GraphicConverter software.

Production and Purification of FNR Antibody.

A 75-aa N-terminal truncated form of the Synechocystis FNR (37 kDa) was expressed in E. coli BL21 (DE3) from a plasmid provided by J. J. van Thor (University of Amesterdam, Amsterdam, The Netherlands) (36). An isopropyl β-d-thiogalactoside-induced E. coli culture was used to purify the FNR to homogeneity as follows. A 50–70% ammonium sulfate precipitation of the crude soluble extract was followed by column chromatography purifications: DE52 (Whatman, Middlesex, UK), Hitrap Q Sepharose, HiLoad Phenyl Sepharose (Amersham Pharmacia), and a final blue Sepharose affinity. The purified FNR was then used to raise antibodies in rabbits. A pure Ig fraction was obtained from the crude sera of rabbits as in ref. 37.

Construction of the Mutagenic Plasmid.

A 318-bp fragment (213 bp upstream to 105 bp downstream of the petH first ATG codon) was amplified from genomic DNA by using primers SB and BS. This fragment was cloned between XbaI and BamHI sites of pBC, creating pSB7. A 2,561-bp BamHI–EcoRV fragment containing the Synechocystis petH gene (710 bp upstream of the ATG to 612 bp downstream of the stop) was amplified from genomic DNA by using primers BE and EB. This fragment was cloned between BamHI and EcoRV sites of pSB7, yielding pSBH. The omega cassette was inserted in the unique BamHI site of pSBH, yielding the mutagenic plasmid pSBHΩ (Fig. 8). Sequences of primers used in this study are listed in Table 1, which is published as supporting information on the PNAS web site.

Site-directed mutagenesis was performed by PCR on a plasmid containing a 570-bp XbaI–MscI fragment. The base transversion in MI6 was created with the mutagenic primer pair IFN and WRN, their overlapping 5′ ends containing a silent mutation eliminating an AlwNI site; the mutation was carried by IFN. Frameshift mutations in FS1 and FS2 were created by cytidine insertion and deletion, respectively, at the AlwNI site. Primers SHF and SHR were used for FS1, and SIF and SIR were used for FS2. Plasmids were sequenced to ensure that no error occurred during the PCR and that the desired mutations were created.

Transformation of Synechocystis PCC 6803.

The WT Synechocystis was transformed by mutagenic plasmids carrying each of the three mutations MI6, FS1, and FS2. Transformants were selected on plates containing 50 μg·ml⁻¹ spectinomycin and 20 mM glucose. Complete segregation was confirmed by PCR and restriction analysis.

Abbreviations

APC

allophycocyanin

FNR

ferredoxin:NADP(H) oxidoreductase

IRES

internal ribosome entry site

PBS

phycobilisome

PBP

phycobiliprotein

PC

phycocyanin.