The gene cphA encoding cyanophycin synthetase was interrupted in Anabaena variabilis ATCC 29413 by insertional mutagenesis. The mutant lacked cyanophycin granules and the polar nodules of heterocysts. The mutant grew as fast as the wild-type irrespective of the nitrogen source at low light intensity whereas growth on N2 was somewhat reduced in high light. It is concluded that cyanophycin metabolism and polar nodules are not essential for aerobic N2 fixation.
mutant of Anabaena 29413 with interrupted cphA gene
sodium dodecyl sulfate-polyacrylamide gel electrophoresis
The branched polypeptide cyanophycin (multi-l-arginyl-poly-l-aspartic acid), a product of non-translational peptide synthesis, is a nitrogen-rich reserve material found in most cyanobacteria (reviewed in [1,2]). The polymer, which is insoluble at physiological pH values, is deposited in the cytoplasm of vegetative cyanobacterial cells in the form of structured granules . Certain filamentous cyanobacteria such as species of Anabaena and Nostoc are able to form specialized cells called heterocysts which are the sites of aerobic N2 fixation. Based on cytochemical staining for arginine  and electron microscopic evidence [5,6], material accumulating in the polar channels of heterocysts, the so-called polar nodules, may also consist of or contain cyanophycin. A recent mutational analysis  of gene argL of Nostoc ellipsosporum, a paralog of gene argC encoding an enzyme of arginine biosynthesis, N-acetylglutamate semialdehyde dehydrogenase, correlates lack of cyanophycin granules with impaired N2 fixation. The identification of gene cphA as the structural gene of cyanophycin synthetase [8,9] makes it possible to address the physiological function of cyanophycin in a more direct way. Here we describe properties of an engineered mutant of Anabaena variabilis ATCC 29413 strain FD (Anabaena 29413), a filamentous, heterocyst-forming cyanobacterium, with an interrupted cphA gene. The mutant lacks cyanophycin and the polar nodules of heterocysts. However, it grows on N2 under aerobic conditions, albeit slower than the wild-type at high light intensity.
2Materials and methods
2.1Strains and culture conditions
Anabaena 29413  was routinely grown at 29°C under continuous illumination with white light in cyanobacterial growth medium of Allen and Arnon (AA-medium)  lacking combined nitrogen. Unless stated otherwise, average light intensities were 40 μE m−2 s−1 for cultures grown in liquid (four-fold dilution of AA-medium) and 15 μE m−2 s−1 for cultures grown on agar (AA-medium containing 1% (w/v) Bacto Agar). Media for recombinant derivatives of Anabaena 29413 contained 50 μg kanamycin ml−1. Liquid cultures were rigorously bubbled with filtered air containing 2% (v/v) CO2. Strains of Escherichia coli were grown in LB medium (LB Broth, LB Agar; Gibco BRL) supplemented, when appropriate, with one or combinations of the following antibiotics (in μg ml−1): chloramphenicol, 25; kanamycin, 50; ampicillin, 50.
2.2Recombinant DNA techniques
Routine DNA manipulations were performed as in Sambrook et al. . Plasmid DNA was isolated with the High Pure Plasmid Isolation kit (Roche Molecular Biochemicals). Polymerase chain reaction (PCR) was performed with the PCR Core kit (Roche Molecular Biochemicals) or Ready-to-go PCR beads (Amersham Pharmacia), and the products purified with High Pure PCR Product Purification kit (Roche Molecular Biochemicals). DNA fragments were recovered from agarose gels with the Qiaex II Gel Extraction kit (Qiagen), and DNA probes labelled with [α-32P]dCTP (Amersham Pharmacia) by random priming (RediprimeII Random Prime Labelling kit from Amersham Pharmacia).
2.3Interposon mutagenesis of gene cphA
A gene library of Anabaena 29413 was constructed from genomic DNA , digested to fragments ranging from 7 to 12 kbp by partial digestion with SauIIIA I, in the λ vector system ZAP Express (Stratagene) according to the instructions of the supplier. The library was screened by plaque hybridization using a 32P-labelled PCR fragment comprising bp 3–1375 of gene cphA (slr2002) of Synechocystis sp. PCC6803 [8,14]. The DNA inserts of five positive clones were excised as pBK-CMV phagemids in host strain E. coli XLORL (Stratagene). By restriction analysis, two clones (Avar15 and Avar17) with overlapping inserts were selected and completely sequenced by the chain-termination method  on both strands (EMBL Data Library, accession number AJ005201). Plasmid DNA of clone Avar17 was digested with PstI and XbaI and a 3.15-kbp fragment was recovered that contained 18 bp of the multiple cloning site of pBK-CMV, the entire coding region of cphA except for the first 14 bp, and 392 bp of the downstream region. It was ligated between the PstI site and the SpeI site (destroyed) of plasmid pRL271 . After partial digestion with SpeI, the kanamycin/neomycin resistance cassette C.K3 , isolated as XbaI fragment from plasmid pRL448 , was ligated into the SpeI sites and the resulting construct used to transform E. coli strain DH5α. Kanamycin-resistant clones containing cassette C.K3 in the internal SpeI site of cphA (Fig. 1) were identified by restriction analysis. One such clone was transferred to Anabaena 29413 by conjugation, and double recombinants were selected using published procedures  on AA-medium containing 5 mM KNO3 as nitrogen source.
Ultrastructural investigations and immunocytochemical visualization of cyanophycin with an antiserum raised against cyanophycin isolated from Synechocystis PCC 6803 was performed as described elsewhere (Stephan, Ruppel and Pistorius, submitted for publication).
Preparation of crude extracts, measurement of cyanophycin synthetase activity, purification of cyanophycin, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) were performed as previously described . For immunoblotting, proteins were transferred electrophoretically to nitrocellulose membranes. For immunodecoration, a rabbit antiserum raised against cyanophycin synthetase from Anabaena 29413 was used. Antigen–antibody complexes were visualized with peroxidase-conjugated protein A (Sigma) and developed with the ECL Western blot detection kit (Amersham Pharmacia). Chlorophyll and protein were determined as in .
3.1Genetic and biochemical characterization of the ΔcphAAva mutant
The genomic region of Anabaena 29413 that has been sequenced (EMBL Data Library accession number AJ005201) covers two genes encoding enzymes of cyanophycin metabolism. Like in Synechocystis sp. PCC 6803 , gene cphA (encoding cyanophycin synthetase) of Anabaena 29413 is preceded by cphB (Fig. 1, bottom). Gene cphB encodes cyanophycinase, a cyanophycin-specific exopeptidase. For knock-out of cyanophycin synthetase, the kanamycin/neomycin-resistance cassette C.K3 was inserted into the unique SpeI site of cphA. The mutation was confirmed by Southern analysis of DNA isolated from the wild-type and the kanamycin-resistant mutant clone (Fig. 1). The sizes of restriction fragments obtained by digestion with either HincII or PmlI/PacI are those predicted for homologous recombination (HincII digest, 2.4 kbp, 0.9 kbp (WT) and 1.95 kbp, 1.6 kbp, 0.9 kbp (mutant); PmlI/PacI digest, 2.3 kbp (WT) and 3.45 kbp (mutant)). The results indicate that all chromosomal copies of cphA of the filamentous cyanobacterium were interrupted. This conclusion is corroborated by the following results: (i) No signal in the 98-kDa region, the size of cyanophycin synthetase, was observed in immunoblots (Fig. 2A). (ii) The mutant lacked cyanophycin (Figs. 2B and 3). (iii) Activity of cyanophycin synthetase was not detectable in extracts of the mutant (not shown). In conclusion, mutant ΔcphAAva is unable to synthesize the polymer.
3.2Mutant ΔcphAAva lacks cyanophycin granules and polar nodules
Growth of Anabaena 29413 in medium reduced in phosphate (reduction from 0.4 to 0.01 mM phosphate with nitrate as N-source) led to substantial cyanophycin granule accumulation in wild-type cells as previously shown for several other cyanobacteria (1,2), while the mutant ΔcphAAva was not able to synthesize cyanophycin granules under such growth conditions (Fig. 3). Moreover, the results of Fig. 4 show that under N2-fixing growth conditions the polar nodule of heterocysts is missing in the mutant. Immunocytochemical investigations with an antiserum raised against cyanophycin gave clear evidence that the polar nodule being present in wild-type heterocysts contains cyanophycin (Fig. 4B).
3.3Growth on N2 or nitrate as N-source
Growth of Anabaena 29413 wild-type and mutant ΔcphAAva was determined in four-fold diluted AA-medium either lacking combined nitrogen or containing nitrate, under continuous illumination. At the low incident light intensity of 40 μE m−2 s−1, the mutant grew, within experimental error, as fast as the wild-type on N2 as well as nitrate (not shown). At 250 μE m−2 s−1, the growth behavior of mutant and wild-type was again not significantly different in the presence of nitrate (Fig. 5A,B). On N2, however, the cyanophycin-less mutant grew significantly slower as long as the cell density, and therefore self-shading, was low (Fig. 5C,D). Under these growth conditions, the filaments of the mutant (13.2±1.2 cells per filament) were shorter than those of the wild-type (31.0±2.8 cells per filament). The heterocysts frequency (heterocysts per total cells) was slightly increased from 5.8±0.2% in the wild-type to 7.4±0.6% in the mutant, respectively. Such an increased heterocysts frequency in cultures with reduced filament length has been reported previously for blended cultures of Anabaena cylindrica, and has been interpreted to indicate that heterocysts inhibit heterocyst formation in their vicinity .
Heterocysts of mutant ΔcphAAva are devoid of polar nodules (Fig. 4). This supports previous cytochemical and structural evidence [4–6] that cyanophycin is a major constituent of these structures. Because of its high nitrogen content, cyanophycin is an almost ideal nitrogen reserve . Besides such a more long-term function, the polymer has been suggested to serve as a dynamic reservoir for fixed nitrogen, especially in heterocysts which have much higher extractable activities of both cyanophycin synthetase and cyanophycinase than vegetative cells ([23,24] and references therein). The result that the Anabaena 29413 wild-type grows faster on N2 than mutant ΔcphAAva when light is not limiting (Fig. 4C,D) is consistent with such a function.
Our results show that cyanophycin is not essential for aerobic N2 fixation by heterocystous cyanobacteria, whereas mutational analysis of gene argC of N. ellipsosporum has implicated such a role . This discrepancy could reflect species-specific differences. However, as the hypothetical argC gene product may function in arginine biosynthesis, and not immediately in cyanophycin metabolism, we consider it likely that disruption of argC has more consequences than just inhibition of cyanophycin accumulation.
This work was supported by a Grant from the Deutsche Forschungsgemeinschaft (Lo 286/6–2) and by the Fonds der Chemischen Industrie.