Newly Identified Cytochrome c Oxidase Operon in the Nitrogen-Fixing Cyanobacterium Anabaena sp. Strain PCC 7120 Specifically Induced in Heterocysts

Abstract

Two operons have been cloned from Anabaena sp. strain PCC 7120 DNA, each of which encodes the three core subunits of distinct mitochondrial-type cytochrome c oxidases. The two operons are only 72 to 85% similar to one another at the nucleotide level in the most conserved subunit. One of these, coxBACII, is induced >20-fold in the middle to late stages of heterocyst differentiation. Analysis of green fluorescent protein reporters indicates that this operon is expressed specifically in proheterocysts and heterocysts. The other operon, coxBACI, is induced only 2.5-fold following nitrogen step-down and is expressed in all cells. Surprisingly, a disruption mutant of coxAII, the gene encoding subunit I of the heterocyst-specific oxidase, grows normally in the absence of combined nitrogen. It is likely that coxBACI and/or two other putative terminal oxidases present in the Anabaena sp. strain PCC 7120 genome are able to compensate for the loss of the heterocyst-specific oxidase in providing ATP for nitrogen fixation and maintaining a low oxygen level in heterocysts.

The filamentous cyanobacterium Anabaena sp. strain PCC 7120 differentiates specialized cells, called heterocysts, at regular intervals when deprived of combined nitrogen. These cells fix nitrogen while the vegetative cells continue to carry out oxygenic photosynthesis. Nitrogen fixation and oxygen-evolving photosynthesis are nominally incompatible processes because nitrogenase, the enzyme that catalyzes the reduction of nitrogen to ammonium, is extremely sensitive to oxygen. Upon exposure to air, nitrogenase from heterocyst-forming cyanobacteria is irreversibly oxidized and precipitates (10, 13, 16). Heterocysts provide the anaerobic environment in which nitrogenase can function. The oxygen-evolving photosystem II complex is inactivated in these cells, and a semipermeable barrier to gases is provided by the heterocyst envelope, which consists of an inner glycolipid layer and an outer polysaccharide layer (29). A 40% higher rate of respiratory activity has been observed in isolated, intact heterocysts compared to that with whole filaments (11). In addition to providing the high ATP level required for nitrogenase function, it has been proposed that an increased rate of oxygen reduction might be one mechanism by which the anaerobic environment in the heterocysts is established and maintained (28). The major terminal oxidase of the electron transport chain in cyanobacteria is a mitochondrial-type cytochrome caa₃ oxidase (2). Thus, cytochrome c oxidase may play a critical role both in dissipating O₂ and in supplying the large ATP requirement of nitrogenase.

An operon encoding subunits I, II, and III of the mitochondrial-type cytochrome caa₃ oxidase has been cloned from several cyanobacterial species (1, 24, 36, 41). In this report, we describe two operons that each encode all three core subunits of a cytochrome c oxidase. Subunit II from each operon contains the copperA (CuA) motif that is a distinctive feature of cytochrome c oxidases. First, we have isolated and sequenced a cytochrome c oxidase operon (coxBACII), the expression of which increases sharply during heterocyst differentiation and is localized specifically to heterocysts and to proheterocysts. A mutant disrupted in the coxAII gene, which encodes subunit I of the heterocyst-specific cytochrome c oxidase, exhibits no growth defects in either the presence or absence of fixed nitrogen. We have also cloned a second cytochrome c oxidase operon, coxBACI, that appears to be the ortholog of the previously described coxBACI operon of Anabaena variabilis (36). The coxBACI operon is expressed in all cells, and its expression is slightly increased in response to nitrogen limitation, although that expression returns to the prelimitation level before mature heterocysts have formed.

Functional redundancy of respiratory oxidases in Anabaena sp. strain PCC 7120 may be able to compensate for mutations in one oxidase operon. Anabaena sp. strain PCC 7120 appears to contain four respiratory oxidase operons: (i) coxBACII (described in this report); (ii) coxBACI (described in this report and in reference 36); (iii) all4023 and all4024 (cydAB), a two-subunit operon defined as a cytochrome D ubiquinol oxidase (23); and (iv) coxIII (found in the completed Anabaena sp. strain PCC 7120 genome [23]). coxIII has been designated as a cytochrome c oxidase, although subunit II of this operon lacks a CuA motif.

MATERIALS AND METHODS

Cloning and plasmid construction.

Restriction enzymes, T4 polymerase, and ligase were obtained from Roche. Primers were synthesized by Life Technologies/Invitrogen. PCRs used Expand High Fidelity Mix, Taq DNA polymerase from Roche or MBI Fermentas, or Pfu polymerase from Stratagene. Plasmid DNA was prepared by the alkaline lysis method (35). All sequencing reactions were done by the University of Chicago CRC DNA Sequencing Facility.

Anabaena sp. strain PCC 7120 coxBII was identified by sequence similarity to known cytochrome c oxidase subunit II genes on a 6-kb subclone of cosmid 59-1-41 from a library previously described (7). The complete sequence of coxBACII was obtained by primer walking and further subcloning of the cosmid insert into Bluescript KS (Stratagene). The transcriptional reporter coxBII::gfp33.1 was constructed by PCR amplification of 0.6 kb upstream of the coxBII start codon with primers ctaCupstr.upper and ctaCupstr.lower, which were designed with SmaI sites at their 5′ ends. The SmaI-digested fragment was cloned into the pAM1956 SmaI site directly upstream of gfp-mut2, which was kindly provided by H. S. Yoon and J. W. Golden (Texas A & M University) (43). pAM1956 carries a neo/kan resistance marker. The resulting plasmid, coxBII::gfp33.1, was checked for amplification errors by sequencing and then was conjugated into wild-type Anabaena sp. strain PCC 7120 (25). Colonies were selected on neomycin sulfate (30 μg/ml).

The Anabaena sp. strain PCC 7120 coxBI sequence was obtained from a PCR product amplified from Anabaena sp. strain PCC 7120 DNA with primers varcoxB1-34 and varcoxA1503, which were derived from the sequence of A. variabilis coxBACI (36). Additional sequence upstream of coxBI was obtained by circularizing an NheI genomic digest and amplifying across the point of ligation with primers varcoxB223 and varcoxBinternal521. The sequence of Anabaena sp. strain PCC 7120 coxAI was found by using the coxBI-coxAI intergenic region sequence, which we obtained, to search the contigs of the Anabaena sp. strain PCC 7120 genome from the Kazusa DNA Research Institute (www.kazusa.or.jp/cyano/index.html) (23). The transcriptional reporter coxBI::gfp42.1 was constructed by amplifying 0.3 kb upstream of the coxBI start codon with primers 7120vegcoxups and 7120vegcoxGTG and cloning the SmaI-digested product into pAM1956, as described above.

The coxAII gene disruption construct pcta167RL277 was made by amplifying a 0.4-kb fragment internal to coxAII with primers ctaknockout.upper and ctaknockout.lower with the enzyme Pfu (Stratagene). This fragment was blunt-end ligated into the NruI site of the vector pRL277 (kindly provided by J. Elhai, University of Richmond; GenBank accession number L05082), which contains the aadA gene conferring streptomycin and spectinomycin resistance (12).

Phylogenetic tree construction.

The nucleotide coding sequences of the genes listed below were aligned in Clustalx (18, 42), and the phylogram was generated with PAUP 4.0 (39). The sequences used in the alignment were the following: Anabaena sp. strain PCC 7120 coxAII (GenBank accession number AF291994; this study); Synechococcus vulcanus ctaD (GenBank accession number D16254 [38, 41]); Synechocystis sp. strain PCC 6803 ctaD (GenBank accession number X53746 [1, 24]); Anabaena sp. strain ATCC29413 (A. variabilis) coxAI (GenBank accession number Z98264 [36]); Anabaena sp. strain PCC 7120 coxAI (this study and reference 23); and Paracoccus denitrificans COI (GenBank accession number X05829 [33]).

Transformation and conjugation.

Plasmids were electroporated into Escherichia coli DH10B (Gibco BRL) with a Bio-Rad gene pulser. E. coli carrying cyanobacterial plasmids was cultured in Luria-Bertani medium containing 50 μg of kanamycin/ml or 100 μg of spectinomycin/ml. Plasmids to be transferred into Anabaena sp. strain PCC 7120 were first transformed into E. coli UC585 by the CaCl₂ method (35) and then were conjugated into Anabaena sp. strain PCC 7120 by biparental mating (25). Positive exconjugants were selected on BG-11 plus 17.6 mM NaNO₃ and 1 mM NaS₂O₃ plates containing 30 μg of neomycin/ml or 2 μg of streptomycin/ml and 2 μg of spectinomycin/ml or in a solution containing liquid BG-11 17.6 mM NaNO₃, 2 μg of streptomycin/ml, and 2 μg of spectinomycin/ml.

Cell culture.

Wild-type Anabaena sp. strain PCC 7120 was maintained on BG-11 N_o (nitrogen-free) plates supplemented with 1 mM NaS₂O₃ (34). Strains containing plasmids with the neo/kan resistance marker were maintained on BG-11 plus 17.6 mM NaNO₃ and 1 mM NaS₂O₃ plates containing 30 μg of neomycin sulfate/ml. Strains containing plasmids with the streptomycin/spectinomycin resistance marker were maintained on BG-11 plus 17.6 mM NaNO₃ and 1 mM NaS₂O₃ plates containing 2 μg of streptomycin/ml and 2 μg of spectinomycin/ml.

Cultures were grown in BG-11 N_o medium or medium supplemented with 17.6 mM NaNO₃ or nitrate plus 1 mM [NH₄]₂S0₄. Small cultures (125 to 500 ml) were grown in a 30 to 33°C incubator gassed with a mixture of air and 2 to 3% CO₂ and illuminated by cool white fluorescent bulbs at 30 to 40 μE/m²/s. Cultures for RNA isolation were grown in a stirred 4-liter bottle illuminated by fluorescent ring bulbs and gassed with the same mixture of air and 2 to 3% CO₂. Culture maintenance and transfer from nitrogen-replete medium to N_o medium were as described previously (22). Cell growth was assessed by measuring the absorbance at 750 nm (37, 40).

Confirmation of mutant genotype by PCR.

The coxAII disruption strains were analyzed by colony PCRs, following the completion of the growth experiment shown in Fig. 7, to confirm the continued presence of the disruption plasmid.

FIG. 7.

Growth of the coxAII disruption (disr.) mutant strains compared with that of wild-type Anabaena sp. strain PCC 7120. (A and B) The growth of the coxAII disruption strains was measured as absorbance at an optical density of 750 nm (OD₇₅₀) (37, 40). Growth in the presence (N⁺) or absence (N_o) of combined nitrogen was the same for the coxAII disruption strains as for wild type.

Southern blotting and hybridization.

Anabaena sp. strain PCC 7120 or A. variabilis (strain ATCC29413, kindly provided by T. Thiel, University of Missouri) cultures were grown in the nitrate medium described above. DNA was prepared according to methods described previously (6, 14). Ten micrograms of genomic DNA from Anabaena sp. strain PCC 7120 or A. variabilis was digested with AsnI or NheI.

Genomic digests were run on a 1× Tris-acetate-EDTA-0.8% agarose gel, which was prepared for blotting according to the GeneScreen Plus protocol (NEN) and transferred in 10× SSC (1× SSC is 0.15 M NaCl plus 0.015 M sodium citrate) to a GeneScreen Plus nylon membrane. DNA was fixed to the membrane as described previously (22). Probes were labeled by random priming with the Ambion Strip-Easy DNA probe synthesis kit or the Roche Random primed DNA labeling kit with [³²P]dCTP or [³²P]dATP from Amersham. The probes were primed from the following fragments. The coxAI probe was from a 0.9-kb fragment within the coding region of A. variabilis coxA, kindly supplied as a pUC118 clone by G. Schmetterer (University of Vienna). The coxBI probe was from a PCR product amplified from A. variabilis genomic DNA with the primers var.coxB.internal.521 and varBint.lower831. The coxBII-coxAII intergenic region probe was from a PCR product amplified from Anabaena sp. strain PCC 7120 DNA with the primers ctaC-Dupper and ctaC-Dlower.

Aqueous hybridization and washes of the blots were performed as described previously (22). Blots were imaged by exposure to a Type BAS-III Fuji PhosphorImager screen for 2 h. The exposed screen was scanned with a Molecular Dynamics Storm 860 PhosphorImager and analyzed with Molecular Dynamics IQMac software.

Northern blotting and hybridization.

At each time, 300 ml was removed from the culture and RNA isolation was performed with the Ambion Totally RNA kit protocol, with modifications described elsewhere (22). RNA concentration was determined by measuring absorbance at 260 nm. A 15-μg loading aliquot of RNA from each sample was ethanol precipitated and resuspended in 5 parts Sigma RNA loading buffer (M5755) to 1 part RNase-free water. Formaldehyde final concentration was 0.95 M, formamide final concentration was 52%, and ethidium bromide final concentration was 42 μg/ml. Samples were run on a 14- by 18-cm gel containing 1% agarose, 1× MESA buffer (Sigma), and 2% formaldehyde and were transferred in 10× SSC to a GeneScreen Plus nylon membrane.

Loading was assessed by scanning baked blots with the blue laser of the Storm 860 PhosphorImager at a photomultiplier voltage of 900 V. Under these conditions the imager detects ethidium bromide-stained rRNA bands (9). The doublet 23S rRNA bands of Anabaena sp. strain PCC 7120 were quantified and used to normalize the ³²P signal from probes to specific mRNAs. Probes were prepared as outlined above for Southern blotting and hybridization. Blots were prehybridized and hybridized at 42°C in 40 ml of Ambion Ultrahyb Hybridization buffer. Blots were imaged as described above for Southern blotting and hybridization.

RT-PCR.

Two-microgram aliquots of total RNA were digested with DNaseI (Roche). Half of each DNased RNA aliquot was reverse transcribed from the coxII RT primer at 50°C using SuperScript II (Lifetech/Invitrogen). One-tenth of each reverse transcription (RT) reaction or each DNased RNA control aliquot was used for PCR with one of the following primer pairs: coxII RT primer/primer 1; coxII RT primer/primer 4; or coxII RT primer/primer 5. The positive control reaction was performed with coxII RT primer/primer 5 and 6 ng of Anabaena sp. strain PCC 7120 genomic DNA as template. The RT-PCRs were performed with Taq polymerase (MBI Fermentas) for 55 cycles with the following cycling protocol: 94°C denaturation, 30s; 50°C annealing, 30s; 72°C polymerization, 90s.

RNA folding calculations.

The RNA folding calculations on the 38-base element located upstream of the coxBACII operon used the mfold program at the website of M. Zuker (http://bioinfo.math.rpi.edu/∼mfold/rna/) (26, 44).

Microscopy.

Cells were pipetted onto 1% agar cushions containing BG11 with or without nitrate for photography. The strain carrying the coxBII::gfp33.1 construct was photographed with 400-speed film with a Contax 167MT camera attached to a Zeiss Axioskop microscope. Images of green fluorescent protein (GFP) fluorescence were taken by illumination with light (450- to 490-nm wavelength) from a Zeiss HBO100W/2 source and photographing emission through a 510-nm-wavelength narrow band pass filter with a 16-s exposure. Red emission from photosynthetic pigments was photographed without the 510-nm-wavelength filter. The strains carrying the promoterless gfp construct pAM1956 and those carrying coxBI::gfp42.1 were imaged with an Axiovision color CCD camera attached to an Axioplan2 Zeiss microscope. The latter images of GFP fluorescence required 10-s exposures with the CCD camera when the GFP was excited at 450 to 490 nm and viewed through the 500- to 550-nm-wavelength filter of the Endow GFP filter set (Chroma).

Nucleotide sequence accession number.

The Genbank accession number for Anabaena sp. strain PCC 7120 coxAII is AF291994.

RESULTS

Identification of the paralog operons coxBACII and coxBACI in Anabaena sp. strain PCC 7120.

While sequencing the region of Anabaena sp. strain PCC 7120 DNA surrounding the patB gene (25; K. M. Jones, W. J. Buikema, and R. Haselkorn, unpublished data), we found a 3.4-kb segment with a high degree of similarity to cytochrome c oxidase operons from a wide range of organisms. This putative operon, containing three open reading frames, was designated coxBACII. A cytochrome c oxidase operon encoding subunits I, II, and III (coxBACI) had previously been identified in the closely related strain A. variabilis (ATCC29413) (36). Comparison of the coxBACII and coxBACI sequences revealed that the sequence identity in the coding region of the highly conserved subunit I (Anabaena sp. strain PCC 7120 coxAII and A. variabilis coxAI) was confined to short gapped segments with an average identity at the nucleotide level of only 85%, much lower than is typical for homologous sequences from these two strains. This led us to believe that A. variabilis coxBACI was not the ortholog of Anabaena sp. strain PCC 7120 coxBACII and that there might be an additional coxBAC operon in Anabaena sp. strain PCC 7120.

To explore this possibility, a Southern blot was made with genomic DNA from Anabaena sp. strain PCC 7120 and A. variabilis. Identical blots (Fig. 1) were probed as described in the legend to Fig. 1. As expected, the coxBI probe detected an A. variabilis AsnI band of 1.9 kb and an NheI band of approximately 2.8 kb (Fig. 1A). This probe also hybridized to bands from the Anabaena sp. strain PCC 7120 genome, indicating that this sequence is also present in this organism. The coxBII-coxAII intergenic region probe bound to an Anabaena sp. strain PCC 7120 AsnI fragment of 3.0 kb and an NheI fragment of approximately 11.0 kb (Fig. 1C). It also detected bands of similar sizes in the A. variabilis genome. The highly conserved coxAI probe produces a strong signal with an A. variabilis AsnI band of 1.94 kb and an NheI band of 1.94 kb (Fig. 1B). It also produces a strong signal with an Anabaena sp. strain PCC 7120 AsnI band of 1.89 kb and an NheI band of 3.8 kb. This probe also weakly hybridized to bands of the same sizes as those detected by the coxBII-coxAII probe.

FIG. 1.

Southern blot of cytochrome c oxidase genes. Genomic DNA from Anabaena sp. strain PCC 7120 or A. variabilis was digested with either AsnI or NheI, electrophoresed, and blotted as described in Materials and Methods. Identical blots were probed with a 0.31-kb PCR product internal to A. variabilis coxBI (A); a 0.9-kb fragment internal to A. variabilis coxAI (B); or a 0.16-kb PCR fragment from the Anabaena sp. strain PCC 7120 coxBII-coxAII intergenic region (C). Numbers on the left indicate molecular size standards in kilobases.

Taken together, these Southern blot data indicate that each species has sequences contained in the coxBACII operon as well as in the coxBACI operon. To corroborate these results, we obtained the sequence of the Anabaena sp. strain PCC 7120 coxBACI operon identified in the Southern blots. This was done by using several primer sets designed from the A. variabilis coxBACI sequence to amplify the operon from Anabaena sp. strain PCC 7120. A 1.5-kb PCR product spanning coxBI and the coxBI-coxAI intergenic region was obtained by using primers varcoxB1-34 and varcoxA1503. The sequence of this fragment was then used to search the Anabaena sp. strain PCC 7120 genome database at the Kazusa DNA Research Institute (23). The entire coxAI sequence was located on contig 72 (C72).

To examine the relationship between the various cytochrome c oxidase operons, we constructed a phylogenetic tree comparing the Anabaena sp. strain PCC 7120 coxAII and coxAI sequences with those of A. variabilis coxAI, Synechococcus vulcanus ctaD, and Synechocystis PCC 6803 ctaD (Fig. 2). The tree demonstrates that Anabaena sp. strain PCC 7120 coxAI and A. variabilis coxAI are within the same group, while Anabaena sp. strain PCC 7120 coxAII is grouped with ctaD from the marine thermophilic cyanobacterium S. vulcanus and away from Anabaena sp. strain PCC 7120 coxAI. When the paralog of subunit I from the putative coxIII operon is included in the tree, it diverges into its own group, completely separate from coxAII and coxAI (data not shown).

FIG. 2.

Phylogenetic tree of cytochrome c oxidase subunit I. The nucleotide coding sequences of cytochrome c oxidase subunit I from the following organisms were aligned in Clustalx (18, 42), and the phylogram was generated with PAUP 4.0 (39): Anabaena sp. strain PCC 7120 coxAII (this study); S. vulcanus ctaD (38, 41); Synechocystis sp. strain PCC 6803 ctaD (1, 24); Anabaena sp. strain ATCC29413 (A. variabilis) coxAI (36); and Anabaena sp. strain PCC 7120 coxAI (this study and Kazusa DNA Research Institute database C72) (23). P. denitrificans COI was included as an outgroup (33). The tree was constructed by the neighbor-joining method, and P. denitrificans COI was used to root the tree. The numbers displayed above the branches are the fraction of base substitutions/site. Beneath the branch points in parentheses are the bootstrap percentages of 1,000 trees derived with Jukes-Cantor correction. A tree derived by using the maximum parsimony method preserved all the branch points of the neighbor-joining tree (data not shown).

Induction of coxBACII and coxBACI during nitrogen starvation.

The previously observed increase in respiratory activity during nitrogen starvation led us to speculate that one or both cytochrome c oxidase operons might be induced during nitrogen starvation and heterocyst formation in Anabaena sp. strain PCC 7120. To test this possibility, Northern blots of RNA prepared from nitrogen-depleted cultures were probed with sequences from either operon. RNA was isolated from both nitrate-grown cultures and from the same cultures following addition of 1 mM [NH₄]₂SO₄ to completely repress heterocyst differentiation. Thirty to 36 h later cells from these cultures were washed and transferred to N_o medium. RNA was isolated from samples taken at the times indicated in Fig. 3.

FIG. 3.

Northern blots of coxBII-coxAII and coxBI mRNA. (A) The upper panel shows two nitrogen starvation experiments for Anabaena sp. strain PCC 7120 probed with the coxBII-coxAII intergenic region. The lower panel shows the bipartite 23S rRNA bands from a scan of the blots prior to hybridization, as described in Materials and Methods. These bands were quantified and used to normalize the phosphor signal for specific mRNAs from that lane (9). (B) The upper panel shows a time course for Anabaena sp. strain PCC 7120 probed with a coxBI internal fragment. The lower panel shows the bipartite 23S rRNA bands of Anabaena sp. strain PCC 7120 scanned and quantified as described above. (C) The graph shows the coxBACII signal in time course 1 (filled squares) and time course 2 (open diamonds) and the coxBACI signal (open circles) normalized to 23S rRNA. A 3.8-kb message hybridizing to the coxBACII-specific probe (coxBII-coxAII) is induced 20- to 25-fold during nitrogen starvation, while a 4.0-kb message hybridizing to the coxBACI-specific probe (coxBI) is induced 2.5-fold.

The induction of the mRNA homologous to the coxBACII-specific probe (coxBII-coxAII) following nitrogen removal is shown in Fig. 3A. In two experiments, a 3.8-kb major band and a 3.1-kb minor band are induced 20- to 25-fold at 18 h after nitrogen removal. The 3.8-kb message (which is large enough to contain the 3.4 kb of three open reading frames of coxBACII) is most abundant between 18 and 24 h. Since mature heterocysts become apparent in wild-type filaments at 21 h, this indicates that the coxBACII message is induced midway through the heterocyst differentiation process. Because of the timing and amplitude of induction of this message, we thought that it might be expressed primarily in the heterocysts and might be involved in supplying the energy requirements of nitrogenase and in dissipating oxygen in these cells. We therefore determined the cell type specificity of its expression with GFP fusions (see below).

Figure 3B shows a blot annealed with the coxBACI-specific probe defined above. A 4.0-kb message is slightly induced after addition of 1 mM [NH₄]₂SO₄ but is then induced another 2.5-fold after the cells are deprived of nitrogen. Figure 3C displays the relative levels of this message and the message detected by the coxBACII-specific probe. The amount of coxBACI transcript peaks 12 h after nitrogen step-down, drops, and rises again from 48 to 72 h. The first peak of this coxBACI message occurs well before the appearance of mature heterocysts at 21 h. It is possible that the later peak coincides with the beginning of a second round of heterocyst differentiation. Since coxBACI is induced rather early in the response to nitrogen deprivation, it may be involved in providing ATP for the differentiation process.

Delimitation of coxBACII transcription start point.

The large size of the coxBACII message (3.8 kb) made it difficult to predict the location of the transcription start point. To determine the approximate 5′ end of the coxBACII message, PCRs with nested primers were performed on RNA that had been reverse transcribed with the coxII RT primer (Fig. 4B). The RNA samples that served as RT template were isolated from cells grown under N_o conditions for 18 h in two separate experiments. The gel shown in Fig. 4A and the annotated sequence shown in Fig. 4B show that primers with sequences 73 to 93 and 290 to 317 bases upstream of the coxBII start codon (primer 1 and primer 4, respectively) produce amplified DNA from the RT-RNA template in PCRs with the coxII RT primer. However, the primer that binds 403 to 433 bases upstream of the coxBII start codon (primer 5) does not yield product from RT-RNA template with the coxII RT primer. This indicates that the start of the 5′-most coxBACII transcript is between 410 and 300 bases upstream of the coxBII start codon.

FIG. 4.

coxBACII transcript start and features of the upstream region. (A) Tris-borate-EDTA-polyacrylamide gel of RT-PCR products of the coxBACII upstream region. Lanes 1 and 14 show a 100-bp ladder (MBI Fermentas); lanes 2 to 5 are PCRs with primer 1 and coxII RT primer (see Fig. 4B). Lanes 6 to 9 are PCRs with primer 4 and coxII RT primer. Lanes 10 to 13 and 15 are PCRs with primer 5 and coxII RT primer. The negative-control PCR template in lanes 2 and 3, 6 and 7, and 10 and 11 was RNA from cells grown for 18 h in N_o medium from two separate cultures. The PCR template in lanes 4 and 5, 8 and 9, and 12 and 13 was 18-h N_o RNA that had been reverse-transcribed with coxII RT primer to produce first-strand cDNA. The positive-control PCR template in lane 15 was Anabaena sp. strain PCC 7120 genomic DNA. (B) Annotated sequence of the coxBII upstream region. The binding sites of the primers in Fig. 4B are displayed. The start codon and Shine-Dalgarno sequence (putative ribosome-binding site) are also shown. Two potential binding sites for the NtcA nitrogen regulatory protein are shaded in light gray. A palindromic 38-mer located 48 to 86 bases upstream of the coxBII start codon is shaded in dark gray. (C) The palindromic 38-mer can be folded into a hairpin structure, where ▵G = −19.7 kcal/mole. A series of nested fragments, starting with a 440-base fragment extending from 440 bases upstream to the start codon, were folded with the same parameters. In all cases the structure with the lowest −▵G placed this 38-mer into the same conformation, while other sequences around it varied considerably in their folding. When a 38-mer of a different sequence but with the same base composition is folded with the same parameters, ▵G = −3.0 kcal/mole.

Spatial expression of coxBACII and coxBACI.

The cellular expression patterns of coxBII and coxBI were determined by transcriptional fusions to gfp-mut2, encoding GFP (8). Nitrate or ammonia-grown cells carrying a coxBII reporter (coxBII::gfp33.1) have no visible GFP expression in liquid culture. The level of background fluorescence from these cells is the same as that from cells containing the promoterless gfp construct pAM1956 (data not shown). In contrast, expression from the coxBII reporter is apparent at 12 to 14 h after nitrogen deprivation in individual cells that are spaced at approximately 10-cell intervals and that appear to be proheterocysts. The photograph shown in Fig. 5A shows mature heterocysts, 42 h after removal of combined nitrogen, expressing a high level of green fluorescence. The photograph in Fig. 5B is of red emission from photosynthetic pigments. The heterocysts are distinguished in Fig. 5B by the loss of pigment fluorescence due to their catabolism of phycobilisomes and chlorophyll. Thus, the increased abundance of coxBACII mRNA seen in the Northern blots is due to expression of this operon in heterocysts.

FIG. 5.

Expression of GFP from the coxBII::gfp reporter is heterocyst specific. (A) A composite image of Anabaena sp. strain PCC 7120 cells carrying the coxBII::gfp33.1 transcriptional fusion after 42 h in N_o medium, photographed under phase contrast in full-spectrum light, combined with the same microscopic field photographed with excitation and emission wavelengths of 450 to 490 nm and 510 nm, respectively. Bright green fluorescence is confined to the heterocysts (arrowheads). The inset shows an expanded view of part of the field showing just the GFP fluorescence image. Two heterocysts are evident (arrowheads). No GFP fluorescence from vegetative cells is visible. (B) Photosynthetic pigment epifluorescence of cells excited at 450 to 490 nm and photographed without the emission filter. Heterocysts are the dim cells along the filament. The bar corresponds to a length of 10 μm.

A transcriptional gfp fusion to coxBI (coxBI::gfp42.1) was also constructed and introduced into Anabaena sp. strain PCC 7120. Filaments carrying this construct exhibit low GFP fluorescence when they are grown with nitrate or with ammonia as a nitrogen source (Fig. 6A). In contrast to that with the coxBII reporter, the level of GFP fluorescence from cells carrying this reporter changed very little during nitrogen starvation. The photographs in Fig. 6B and C show filaments at 12 h after transfer to N_o medium. Proheterocysts are already visible (Fig. 6C, arrowheads). The GFP fluorescence from the proheterocysts is hardly brighter than that from the adjacent vegetative cells. Filaments carrying the promoterless gfp construct pAM1956 did not produce a fluorescent signal under the same conditions (data not shown). Thus, the coxBI reporter is expressed faintly in all cells, indicating that it is constitutively transcribed at a low level and that it is not heterocyst specific.

FIG. 6.

Expression of GFP from the coxBI::gfp reporter. (A) Cells carrying the coxBI::gfp transcriptional fusion and grown with 17.6 mM NaNO₃ plus 1 mM [NH₄]₂SO₄ as nitrogen sources were imaged by fluorescent excitation and emission of GFP through a 500- to 550-nm-wavelength filter. Low level, constitutive expression is visible along the filament. (B) The same strain 12 h after removal of combined nitrogen from the medium, viewed with 450- to 490-nm-wavelength excitation of GFP (B) and by phase contrast (C). Arrowheads point to proheterocysts. The bar corresponds to a length of 10 μm.

Phenotype of a disruption mutation in the coxAII gene.

The observation that the coxBII::gfp fusion is expressed only in the heterocysts of filaments grown in N_o liquid medium suggested that the coxBACII operon might encode a cytochrome c oxidase important for heterocyst maintenance and cell growth under N_o conditions (Fig. 7). To test this hypothesis, we created a disruption in coxAII. A 0.4-kb fragment internal to coxAII was inserted into the vector pRL277, which carries the aadA gene conferring streptomycin/spectinomycin resistance (12). This construct was conjugated into Anabaena sp. strain PCC 7120, and strains carrying the insertion plasmid were selected as described in Materials and Methods.

The mutant strains, all of which were confirmed by PCR to carry the disruption plasmid in coxAII, grow normally in the presence or in the absence of combined nitrogen at all stages of growth (Fig. 6A and B). The mutant cells are normal in appearance and the filaments are of normal length. In N_o media, heterocyst formation and pattern of the coxAII mutant strains are indistinguishable from those of the wild type (data not shown).

DISCUSSION

Two cytochrome c oxidases with different temporal and spatial expression patterns have been detected in the genome of Anabaena sp. strain PCC 7120 by Southern analysis and PCR. Phylogenetic analysis of the two copies of subunit I (coxAII and coxAI) indicate that each is part of a different subgroup of cyanobacterial cytochrome c oxidases. The heterocyst-induced coxAII gene is closely related to ctaD from the marine unicellular cyanobacterium S. vulcanus, while the constitutive coxAI gene is more closely related to ctaD from Synechocystis PCC 6803 than to the Anabaena sp. strain PCC 7120 heterocyst-specific coxAII. The coxAIII gene of Anabaena sp. strain PCC 7120 is not closely related to either the coxAII or the coxAI group (data not shown).

Analysis of the sequence of the two Anabaena sp. strain PCC 7120 cytochrome c oxidase operons, coxBACI and coxBACII, indicates that both encode proteins containing conserved residues known to be required for cytochrome c oxidase function. First, both contain a conserved CuA site. Cytochrome caa₃ oxidases communicate with the electron transport chain through an interaction between the solvent-exposed CuA site of subunit II and ferrocytochrome c (27). The recent determination of the crystal structure of the P. denitrificans enzyme allowed the identification of the residues required to bind the two copper atoms (20, 30). A comparison of the amino acid sequence of both coxBII and coxBI open reading frames from Anabaena sp. strain PCC 7120 with that of P. denitrificans subunit II (COII) indicates that both Anabaena sp. strain PCC 7120 sequences possess a CuA site defined by the H-X₅₂-C-X-E-X-C-X₃-H-X₂-M motif (15, 27). In contrast, subunit II of the coxIII operon does not have the critical residues of the CuA site. Second, subunit I from coxAII and coxAI each contains three conserved motifs. In order to function as an oxidase, subunit I must have binding sites for the heme moieties a and a₃ and a copperB site (CuB). Electrons are delivered to subunit I at heme a and are transferred from heme a to the oxygen binding binuclear site composed of heme a₃ and the CuB. The crystal structure of P. denitrificans subunit I (COI) demonstrates that binding of heme a occurs at H78 and H397, heme a₃ at H395, and copper at H260, H309, and H310 (15). The open reading frame of coxAI contains histidines in similar positions H83 and H396; H394; and H259, H308, and H309, while coxAII has similarly spaced histidines in the slightly shifted positions H92 and H405; H403; and H268, H317, and H318.

The coxBACI genes appear to form an operon that is consistent in size with the 4.0-kb message detected with a coxBI probe, suggesting that this transcript is a polycistronic mRNA transcribed from these genes. This message is present at a low level under all conditions tested but is induced 2.5-fold by 12 h after nitrogen limitation. The amount of message has dropped almost to the preinduction level by the time mature heterocysts have formed at 24 h after removal of nitrogen. A subsequent induction at 72 h may coincide with a second round of heterocyst differentiation. GFP expression from a transcriptional fusion of the coxBI upstream elements to gfp is found in both heterocysts and vegetative cells. There are several sequences upstream of the coxBI start codon that might define −35 and −10 promoter elements.

The coxBACII genes also appear to form an operon. Expression of a 3.8-kb message detected by a coxBII-coxAII intergenic region probe is induced >20-fold at 18 to 24 h after removal of nitrogen, which is relatively late in the heterocyst differentiation process. The expression of a gfp reporter fused to the coxBII upstream elements is localized specifically to proheterocysts 12 to 14 h after nitrogen step-down. A 20-fold induction averaged over the total culture, which actually occurs only in the 10% of cells that differentiate, means that the increase in expression in proheterocysts is on the order of 200-fold. The message levels fall after 18 to 24 h, during the time that mature heterocysts form.

The 5′ end of the longest detectable message from the coxBACII operon is at least 300 bases upstream of the coxBII start codon and may extend as far as 410 bases. Two potential binding sites for the NtcA nitrogen regulatory protein are located upstream of coxBACII. One, composed of the sequence TGTA(N)₈TACC, is located 423 to 407 bases upstream of the start codon of coxBII, while the other is TGT(N)₁₀ACA and is 360 to 345 bases upstream of the start codon (17, 21). Either or both of these sites could regulate coxBACII transcription.

Another feature of the coxBACII upstream region is a 38-base imperfect palindrome located 86 to 48 bp upstream of the coxBII start codon (Fig. 4C). This palindrome has the potential to form an RNA stem-loop structure. The calculated free energy (ΔG) of folding of this RNA 38-mer is −19.7 kcal/mol, while the ΔG of folding of a randomly generated 38-mer of the same base composition is −3 kcal/mol (26, 44). Sequences with similarity to this 38-base element are found at several loci in the Anabaena sp. strain PCC 7120 genome (23, 32) but are not found upstream of coxBACI. A consensus sequence derived by comparing the 18-most similar sequences (18, 42), G(N)₂(G)₃(N)₃(C)₃(N)₂C, appears 27 times in the genome and flanks the 5′ end of 22 open reading frames (CodonUse v. 3.5.5) (www.brena.uni-koeln.de) (3-5, 19, 31). Eleven open reading frames have the element in the 3′ flanking region. The element occurs within 7 open reading frames. This element in the upstream flanking region of coxBII is presently under analysis to determine if it is required for normal expression of the coxBACII message.

The sharp increase in coxBACII message levels in middle to late stages of heterocyst differentiation, together with its expression specifically in heterocysts, lead us to speculate that a cytochrome c oxidase encoded by this operon may at least partially account for the increase in respiratory O₂ uptake that has been observed previously in isolated heterocysts (11). However, mutants disrupted in coxAII, which encodes subunit I of cytochrome c oxidase, have no defect in heterocyst formation or in growth in the absence of combined nitrogen. It is likely that in the absence of a functional coxBACII, one or more of the other terminal oxidases of Anabaena sp. strain PCC 7120 is able to compensate. This is consistent with the phenotype of an A. variabilis coxAI mutant which experiences a significant drop in cytochrome c oxidase activity relative to that of the wild type under some culture conditions, but it has no growth defect (36). Despite the reduced level of cytochrome c oxidase activity, this coxAI mutant also has a near-wild-type level of respiratory oxygen consumption, implying that respiratory oxidases that do not interact with cytochrome c can compensate for coxAI (36). The Anabaena sp. strain PCC 7120 genome has, in addition to coxBACII and coxBACI, a two-subunit, putative cytochrome D ubiquinol oxidase operon (cydAB) and an operon of between two and five subunits that has been designated coxIII. Absence of the residues required to form a CuA site in coxBIII suggests that this enzyme may not interact with cytochrome c.