Heterocyst-forming Cyanobacterium Anabaena Research Articles

Inactivation of agmatinase expressed in vegetative cells alters arginine catabolism and prevents diazotrophic growth in the heterocyst-forming cyanobacterium Anabaena.

Arginine decarboxylase produces agmatine, and arginase and agmatinase are ureohydrolases that catalyze the production of ornithine and putrescine from arginine and agmatine, respectively, releasing urea. In the genome of the filamentous, heterocyst-forming cyanobacterium Anabaena sp. strain PCC 7120, ORF alr2310 putatively encodes an ureohydrolase. Cells of Anabaena supplemented with [14C]arginine took up and catabolized this amino acid generating a set of labeled amino acids that included ornithine, proline, and glutamate. In an alr2310 deletion mutant, an agmatine spot appeared and labeled glutamate increased with respect to the wild type, suggesting that Alr2310 is an agmatinase rather than an arginase. As determined in cell-free extracts, agmatinase activity could be detected in the wild type but not in the mutant. Thus, alr2310 is the Anabaena speB gene encoding agmatinase. The Δalr2310 mutant accumulated large amounts of cyanophycin granule polypeptide, lacked nitrogenase activity, and did not grow diazotrophically. Growth tests in solid media showed that agmatine is inhibitory for Anabaena, especially under diazotrophic conditions, suggesting that growth of the mutant is inhibited by non-metabolized agmatine. Measurements of incorporation of radioactivity from [14C]leucine into macromolecules showed, however, a limited inhibition of protein synthesis in the Δalr2310 mutant. Analysis of an Anabaena strain producing an Alr2310-GFP (green fluorescent protein) fusion showed expression in vegetative cells but much less in heterocysts, implying compartmentalization of the arginine decarboxylation pathway in the diazotrophic filaments of this heterocyst-forming cyanobacterium.

Photochemical Properties of a Novel Cyanobacteriochrome from Anabaena sp. PCC7120

Cyanobacteriochromes are phytochrome homologues in cyanobacteria that act as photoreceptor sensors. We report the photochemistry of All4261 GAF2, a novel cyanobacteriochrome from the heterocyst-forming cyanobacterium Anabaena sp. PCC 7120. All4261 contains four tandemly arranged GAF domains. The respective chromophore domains were co-expressed in Escherichia coli with the genes for phycocyanobilin biosynthesis enzymes, HO1, and PcyA. The resulting proteins were analyzed by zinc-induced fluorescence, UV-Vis absorption and emission fluorescence spectra. Only All4261 GAF2 binded chromophore covalently, having zinc-induced fluorescence band in SDS-PAGE gel with the zinc acetate solution existed. Absorption spectra analysis showed that All4261 GAF2 had absorption peaks at 340 nm and 590 nm, failing to show photoreversibility. Fluorescence spectra revealed that All4261 GAF2 had a fluorescence emission peak at 645 nm, with high fluorescence quantum yield and molar extinction coefficient.

ChIP analysis unravels an exceptionally wide distribution of DNA binding sites for the NtcA transcription factor in a heterocyst-forming cyanobacterium

BackgroundThe CRP-family transcription factor NtcA, universally found in cyanobacteria, was initially discovered as a regulator operating N control. It responds to the N regime signaled by the internal 2-oxoglutarate levels, an indicator of the C to N balance of the cells. Canonical NtcA-activated promoters bear an NtcA-consensus binding site (GTAN8TAC) centered at about 41.5 nucleotides upstream from the transcription start point. In strains of the Anabaena/Nostoc genera NtcA is pivotal for the differentiation of heterocysts in response to N stress.ResultsIn this study, we have used chromatin immunoprecipitation followed by high-throughput sequencing to identify the whole catalog of NtcA-binding sites in cells of the filamentous, heterocyst-forming cyanobacterium Anabaena sp. PCC 7120 three hours after the withdrawal of combined N. NtcA has been found to bind to 2,424 DNA regions in the genome of Anabaena, which have been ascribed to 2,153 genes. Interestingly, only a small proportion of those genes are involved in N assimilation and metabolism, and 65% of the binding regions were located intragenically.ConclusionsThe distribution of NtcA-binding sites identified here reveals the largest bacterial regulon described to date. Our results show that NtcA has a much wider role in the physiology of the cell than it has been previously thought, acting both as a global transcriptional regulator and possibly also as a factor influencing the superstructure of the chromosome (and plasmids).

The Outer Membrane TolC-like Channel HgdD Is Part of Tripartite Resistance-Nodulation-Cell Division (RND) Efflux Systems Conferring Multiple-drug Resistance in the Cyanobacterium Anabaena sp. PCC7120

The TolC-like protein HgdD of the filamentous, heterocyst-forming cyanobacterium Anabaena sp. PCC 7120 is part of multiple three-component "AB-D" systems spanning the inner and outer membranes and is involved in secretion of various compounds, including lipids, metabolites, antibiotics, and proteins. Several components of HgdD-dependent tripartite transport systems have been identified, but the diversity of inner membrane energizing systems is still unknown. Here we identified six putative resistance-nodulation-cell division (RND) type factors. Four of them are expressed during late exponential and stationary growth phase under normal growth conditions, whereas the other two are induced upon incubation with erythromycin or ethidium bromide. The constitutively expressed RND component Alr4267 has an atypical predicted topology, and a mutant strain (I-alr4267) shows a reduction in the content of monogalactosyldiacylglycerol as well as an altered filament shape. An insertion mutant of the ethidium bromide-induced all7631 did not show any significant phenotypic alteration under the conditions tested. Mutants of the constitutively expressed all3143 and alr1656 exhibited a Fox(-) phenotype. The phenotype of the insertion mutant I-all3143 parallels that of the I-hgdD mutant with respect to antibiotic sensitivity, lipid profile, and ethidium efflux. In addition, expression of the RND genes all3143 and all3144 partially complements the capability of Escherichia coli ΔacrAB to transport ethidium. We postulate that the RND transporter All3143 and the predicted membrane fusion protein All3144, as homologs of E. coli AcrB and AcrA, respectively, are major players for antibiotic resistance in Anabaena sp. PCC 7120.

Cluster of Genes That Encode Positive and Negative Elements Influencing Filament Length in a Heterocyst-Forming Cyanobacterium

The filamentous, heterocyst-forming cyanobacteria perform oxygenic photosynthesis in vegetative cells and nitrogen fixation in heterocysts, and their filaments can be hundreds of cells long. In the model heterocyst-forming cyanobacterium Anabaena sp. strain PCC 7120, the genes in the fraC-fraD-fraE operon are required for filament integrity mainly under conditions of nitrogen deprivation. The fraC operon transcript partially overlaps gene all2395, which lies in the opposite DNA strand and ends 1 bp beyond fraE. Gene all2395 produces transcripts of 1.35 kb (major transcript) and 2.2 kb (minor transcript) that overlap fraE and whose expression is dependent on the N-control transcription factor NtcA. Insertion of a gene cassette containing transcriptional terminators between fraE and all2395 prevented production of the antisense RNAs and resulted in an increased length of the cyanobacterial filaments. Deletion of all2395 resulted in a larger increase of filament length and in impaired growth, mainly under N2-fixing conditions and specifically on solid medium. We denote all2395 the fraF gene, which encodes a protein restricting filament length. A FraF-green fluorescent protein (GFP) fusion protein accumulated significantly in heterocysts. Similar to some heterocyst differentiation-related proteins such as HglK, HetL, and PatL, FraF is a pentapeptide repeat protein. We conclude that the fraC-fraD-fraE←fraF gene cluster (where the arrow indicates a change in orientation), in which cis antisense RNAs are produced, regulates morphology by encoding proteins that influence positively (FraC, FraD, FraE) or negatively (FraF) the length of the filament mainly under conditions of nitrogen deprivation. This gene cluster is often conserved in heterocyst-forming cyanobacteria.

Exploring the size limit of protein diffusion through the periplasm in cyanobacterium Anabaena sp. PCC 7120 using the 13 kDa iLOV fluorescent protein

In the filamentous heterocyst-forming cyanobacterium Anabaena PCC 7120, vegetative cells and heterocysts are interdependent on each other and engaged in exchanges of metabolites for survival when grown under diazotrophic conditions. In this organism, the periplasm appears to be continuous along each filament, with a shared outer membrane; however, barriers exist preventing free diffusion of the fluorescent protein GFP (27 kDa) targeted into the periplasmic space. Here we expressed a smaller fluorescent protein iLOV (∼13 kDa) fused to the All3333 (a putative homologue of NrtA) signal sequence corresponding to those recognized by the TAT protein translocation system, which exports iLOV to the periplasm of either heterocysts or vegetative cells. Fluorescence microscopy and immunoblot analysis indicated that the iLOV protein is translocated into the periplasm of the producing cell and properly processed, but does not diffuse to neighboring cells via the periplasm. Thus, periplasmic barriers appear to block diffusion of molecules with a size of 13 kDa, the minimum size tested thus far. Assuming that the physical barrier is the peptidoglycan sacculus, its pores might allow diffusion of molecules within the size range between the PatS pentapeptide and iLOV, thus between 0.53 kDa and 13 kDa.

The TolC-like Protein HgdD of the Cyanobacterium Anabaena sp. PCC 7120 Is Involved in Secondary Metabolite Export and Antibiotic Resistance

The role of TolC has largely been explored in proteobacteria, where it functions as a metabolite and protein exporter. In contrast, little research has been carried out on the function of cyanobacterial homologues, and as a consequence, not much is known about the mechanism of cyanobacterial antibiotic uptake and metabolite secretion in general. It has been suggested that the TolC-like homologue of the filamentous, heterocyst-forming cyanobacterium Anabaena sp. PCC 7120, termed heterocyst glycolipid deposition protein D (HgdD), is involved in both protein and lipid secretion. To describe its function in secondary metabolite secretion, we established a system to measure the uptake of antibiotics based on the fluorescent molecule ethidium bromide. We analyzed the rate of porin-dependent metabolite uptake and confirmed the functional relation between detoxification and the action of HgdD. Moreover, we identified two major facilitator superfamily proteins that are involved in this process. It appears that anaOmp85 (Alr2269) is not required for insertion or assembly of HgdD, because an alr2269 mutant does not exhibit a phenotype similar to the hgdD mutant. Thus, we could assign components of the metabolite efflux system and describe parameters of detoxification by Anabaena sp. PCC 7120.

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

In the model, heterocyst-forming cyanobacterium Anabaena sp. PCC 7120, gene cluster alr2877-alr2880, which encodes an ABC-type transport system, was induced under conditions of carbon limitation and its inactivation impaired the uptake of bicarbonate. Thus, this gene cluster encodes a Cmp bicarbonate transporter. ORF all0862, encoding a LysR-type transcriptional regulator, was expressed under carbon limitation and at higher levels in the absence than in the presence of combined nitrogen, with a positive effect of the N-control transcription factor NtcA. all0862 was expressed from two putative transcription start sites located 164 and 64 bp upstream from the gene respectively. The latter was induced under carbon limitation and was dependent on positive autoregulation by All0862. All0862 was required for the induction of the Cmp bicarbonate transporter, thus representing a CmpR regulator of Anabaena sp. These results show a novel mode of co-regulation by C and N availability through the concerted action of N- and C-responsive transcription factors.

The components of the putative iron transport system in the cyanobacterium Anabaena sp. PCC 7120

Iron uptake in Gram-negative bacteria involves four distinct steps: (i) siderophore synthesis, (ii) siderophore secretion into the extracellular space, (iii) iron chelation by the siderophores, and (iv) siderophore/iron uptake via complexes in the outer membrane and the intermembrane space as well as in the plasma membrane. This process is well characterized for some proteobacterial systems, but largely unexplored and scarcely investigated in cyanobacteria such as the heterocyst-forming cyanobacterium Anabaena sp. PCC 7120. Two putative siderophore synthesis clusters have been recently identified in this cyanobacterium. In addition, the export system for the main siderophore, schizokinen, secreted by Anabaena sp. PCC 7120 was described as well as the outer membrane transporter for its import from the extracellular space. We present the identification of components of three additional systems involved in siderophore-mediated iron uptake under iron-limiting conditions, namely TonB3, the ExbB3/ExbD3 and the Fhu systems. The transcription level of these genes is elevated under iron limitations and decreased under excess iron, while the expression levels of other members of these gene families and systems are impacted in distinct ways by other environmental conditions. Mutants of the tonB3, exbB3/exbD3 and fhu genes show an iron starvation phenotype. Thus, Anabaena sp. has a similar, yet distinct system for siderophore-dependent iron uptake compared with other proteobacteria.

Influence of Nano Nutrients on Heterocyst-Forming Cyanobacterium Anabaena ambigua Rao

The present study is deals with the influence of nanoparticles on the growth of heterocyst-forming cyanobacterium Anabaena ambigua Rao [A100]. The nano molybdenum and iron particles have been synthesized respectively from molybdenum trioxide (MoO3) and iron chloride (FeCl3) employing sodium borohydride (NaBH4) as a reducing agent and characterized comprehensively. BG11 (N−) media containing micronutrients like molybdenum and iron were replaced respectively by different concentrations of nano molybdenum [BG11 (N−Mo− + nano Mo)] (0, 5, 10, 15, 20, 25, 40, 60, 80, and 100%) and nano iron [BG11 (N−Fe− + nano Fe)] (0, 0.1, 0.2, 0.3, 0.4, 0.5, 1, 2, 3, 4, 5, 10, 15, 20, 25, 40, 60, 80, and 100%) particles for the cultivation. The effect of nano nutrients on cyanobacterial growth parameters like heterocyst formation, chlorophyll content, and protein content was analyzed. The obtained results showed that the high concentrations of nano molybdenum induce heterocyst formation, increase protein content, and decrease chlorophyll content when compared to the control, whereas the nano iron suppresses the growth of cyanobacteria Anabaena ambigua Rao [A 100].

FtsZ degradation in the cyanobacterium Anabaena sp. strain PCC 7120

In prokaryotes, cell division is normally achieved by binary fission, and the key player FtsZ is considered essential for the complete process. In cyanobacteria, much remains unknown about several aspects of cell division, including the identity and mechanism of the various components involved in the division process. Here, we report results obtained from a search of the players implicated in cell division, directly associating to FtsZ in the filamentous, heterocyst-forming cyanobacterium Anabaena sp. PCC 7120. Histidine tag pull-downs were used to address this question. However, the main observation was that FtsZ is a target of proteolysis. Experiments using various cell-free extracts, an unrelated protein, and protein blot analyses further supported the idea that FtsZ is proteolytically cleaved in a specific manner. In addition, we show evidence that both FtsZ termini seem to be equally prone to proteolysis. Taken together, our data suggest the presence of an unknown player in cyanobacterial cell division, opening up the possibility to investigate novel mechanisms to control cell division in Anabaena PCC 7120.

The interplay between siderophore secretion and coupled iron and copper transport in the heterocyst-forming cyanobacterium Anabaena sp. PCC 7120

Iron uptake is essential for Gram-negative bacteria including cyanobacteria. In cyanobacteria, however, the iron demand is higher than in proteobacteria due to the function of iron as a cofactor in photosynthesis and nitrogen fixation, but our understanding of iron uptake by cyanobacteria stands behind the knowledge in proteobacteria. Here, two genes involved in this process in the heterocyst-forming cyanobacterium Anabaena sp. PCC 7120 were identified. ORF all4025 encodes SchE, a putative cytoplasmic membrane-localized transporter involved in TolC-dependent siderophore secretion. Inactivation of schE resulted in an enhanced sensitivity to high metal concentrations and decreased secretion of hydroxamate-type siderophores. ORF all4026 encodes a predicted outer membrane-localized TonB-dependent iron transporter, IacT. Inactivation of iacT resulted in decreased sensitivity to elevated iron and copper levels. Expression of iacT from the artificial trc promoter (P trc ) resulted in sensitization against tested metals. Further analysis showed that iron and copper effects are synergistic because a decreased supply of iron induced a significant decrease of copper levels in the iacT insertion mutant but an increase of those levels in the strain carrying P trc - iacT. Our results unravel a link between iron and copper homeostasis in Anabaena sp. PCC 7120.

Negative Regulation of Expression of the Nitrate AssimilationnirAOperon in the Heterocyst-Forming CyanobacteriumAnabaenasp. Strain PCC 7120

In the filamentous, heterocyst-forming cyanobacterium Anabaena sp. strain PCC 7120, expression of the nitrate assimilation nirA operon takes place in the absence of ammonium and the presence of nitrate or nitrite. Several positive-action proteins that are required for expression of the nirA operon have been identified. Whereas NtcA and NtcB exert their action by direct binding to the nirA operon promoter, CnaT acts by an as yet unknown mechanism. In the genome of this cyanobacterium, open reading frame (ORF) all0605 (the nirB gene) is found between the nirA (encoding nitrite reductase) and ntcB genes. A nirB mutant was able to grow at the expense of nitrate as a nitrogen source and showed abnormally high levels of nirA operon mRNA both in the presence and in the absence of nitrate. This mutant showed increased nitrate reductase activity but decreased nitrite reductase activity, an imbalance that resulted in excretion of nitrite, which accumulated in the extracellular medium, when the nirB mutant was grown in the presence of nitrate. A nirA in-frame deletion mutant also showed a phenotype of increased expression of the nirA operon in the absence of ammonium, independent of the presence of nitrate in the medium. Both NirB and NirA are therefore needed to keep low levels of expression of the nirA operon in the absence of an inducer. Because NirB is also needed to attain high levels of nitrite reductase activity, NirA appears to be a negative element in the nitrate regulation of expression of the nirA operon in Anabaena sp. strain PCC 7120.

Distribution of heterocyst glycolipids in cyanobacteria

Thirty-four axenic strains of cyanobacteria were analysed for their glycolipid content using high performance liquid chromatography coupled to electrospray ionisation tandem mass spectrometry (HPLC/ESI-MS 2). Species of the families Nostocaceae and Rivulariaceae, capable of biosynthesising heterocysts, contained a suite of glycolipids consisting of sugar moieties glycosidically bound to long-chain diols, triols, keto-ols and keto-diols. The aglycone moiety consisted of C 26 or C 28 carbon-chains with hydroxyl groups at the C-3, ω-1 or ω-3 positions. Keto-ols and keto-diols contained their carbonyl functionalities likely at the C-3 position. These compounds were absent in all analysed unicellular and filamentous non-heterocystous cyanobacteria and in the heterocyst-forming cyanobacterium Anabaena CCY9922 grown in the presence of combined nitrogen, supporting the idea that the long-chain glycolipids are an important and unique structural component of the heterocyst cell envelope. The glycolipids 1-( O-hexose)-3,25-hexacosanediol and 1-( O-hexose)-3-keto-25-hexacosanol were ubiquitously distributed in species of the family Nostocaceae. 1-( O-hexose)-3,25,27-octacosanetriol and 1-( O-hexose)-3-keto-25,27-octacosanediol were dominant in members of the Calothrix genus, while traces of those compounds were detected only in one species of the Nostocaceae family. Their distribution in heterocystous cyanobacteria suggests a chemotaxonomic relevance that might allow distinguishing between species of different genera. Culture experiments indicate that the amount of keto-ols and keto-diols decreases relatively to their corresponding diols and triols counterparts with increasing temperature. Possibly, this is an adaptation to optimise the cell wall gas permeability, preventing inactivation of the oxygen-sensitive nitrogenase while allowing the highest diffusion of atmospheric dinitrogen into the heterocyst.

Role of NtcA, a cyanobacterial global nitrogen regulator, in the regulation of sucrose metabolism gene expression in Anabaena sp. PCC 7120

In the heterocyst-forming cyanobacterium Anabaena sp. PCC 7120 (also known as Nostoc sp. PCC 7120), it has been shown that spsB and susA, the genes coding for proteins related to sucrose synthesis and cleavage, respectively, exhibit converse expression regarding the nitrogen source. In the nitrogen-fixing filament, spsB expression is mostly localized to the heterocysts and susA is only expressed in vegetative cells. The aim of this work was to investigate the participation of NtcA, a global nitrogen regulator that operates in cyanobacteria, in the regulation of sucrose metabolism genes in Anabaena sp. PCC 7120. The induction of spsB expression observed in the filaments upon combined-nitrogen depletion was abolished in an NtcA-deficient mutant. In vitro experiments showed that NtcA binds specifically but with different affinities to two sites in the spsB promoter region. When susA expression was analyzed after a combined-nitrogen starvation, the levels of mRNA, polypeptide and activity increased in the mutant in comparison with the wild-type strain. Also, NtcA interacted with one site in the promoter region of susA. We conclude that sucrose metabolism is coordinated at the transcriptional level with nitrogen metabolism, suggesting a global metabolism regulating role for NtcA.

The amt gene cluster of the heterocyst-forming cyanobacterium Anabaena sp. strain PCC 7120.

The genome of the heterocyst-forming cyanobacterium Anabaena sp. strain PCC 7120 bears a gene cluster including three amt genes that, based on homology of their protein products, we designate amt4, amt1, and amtB. Expression of the three genes took place upon ammonium withdrawal in combined nitrogen-free medium and was NtcA dependent. The genes were transcribed independently, but an amt4-amt1 dicistronic transcript was also produced, and expression was highest for the amt1 gene. A mutant with the whole amt region removed could grow under laboratory conditions using ammonium, nitrate, or dinitrogen as the nitrogen source.

A Novel Photoactive GAF Domain of Cyanobacteriochrome AnPixJ That Shows Reversible Green/Red Photoconversion

We report the discovery of a novel cyanobacteriochrome, the green/red photoreceptor AnPixJ (All1069), isolated from the heterocyst-forming cyanobacterium Anabaena ( Nostoc) sp. PCC 7120. Cyanobacteriochromes are a recently emerging tetrapyrrole-based photoreceptor superfamily that are distantly related to the conventional red/far-red photoreceptor phytochromes (Phys). The chromophore-binding domains of AnPixJ produced in cyanobacterial and Escherichia coli cells both showed a reversible and full photoconversion between a green-absorbing form (λ max = 543 nm) and a red-absorbing form (λ max = 648 nm). Denaturation analysis revealed that the green-absorbing form and the red-absorbing form covalently ligated phycocyanobilin with E-configuration and Z-configuration at the C15 C16 double bond, respectively. Time-resolved spectral analysis showed the formation of the first intermediate state peaking at 680 nm from the dark-stable red-absorbing form. This step resembles the first photoconversion step from the red-absorbing form to the red-shifted lumi-R intermediate state of the Phys. These results suggest that the Pr of AnPixJ is almost equivalent to that of the Phys and starts a primary photoreaction with Z-to- E isomerization in a mechanism similar to that in the Phys, but is finally photoconverted to the unique green-absorbing form.

All4312, an NtcA-regulated two-component response regulator inAnabaenasp. strain PCC 7120

All4312, encoded by open reading frame all4312 in the genome of the heterocyst-forming cyanobacterium Anabaena sp. strain PCC 7120, exhibits a CheY-like receiver domain and an output domain similar to that of OmpR, characteristic of two-component response regulators. Expression of all4312 was directly regulated by NtcA, the global transcriptional regulator of nitrogen assimilation in cyanobacteria. Features characteristic of NtcA-activated promoters were also found upstream from genes encoding All4312 homologues in several other cyanobacterial genomes. Expression of all4312 was however unaffected in a mutant of hetR, which encodes a regulator triggering heterocyst development. The function of All4312 may be related to the cellular response to nitrogen deprivation.

Nitrogen-regulated Genes for the Metabolism of Cyanophycin, a Bacterial Nitrogen Reserve Polymer: EXPRESSION AND MUTATIONAL ANALYSIS OF TWO CYANOPHYCIN SYNTHETASE AND CYANOPHYCINASE GENE CLUSTERS IN THE HETEROCYST-FORMING CYANOBACTERIUM ANABAENA SP. PCC 7120

Two gene clusters each encoding the cyanophycin-metabolism enzymes cyanophycin synthetase and cyanophycinase are found in the heterocyst-forming cyanobacterium Anabaena sp. PCC 7120. In cluster cph1, the genes cphB1 and cphA1 were expressed in media containing ammonium, nitrate, or N(2) as nitrogen sources, but expression was higher in the absence of combined nitrogen taking place both in vegetative cells and heterocysts. Both genes were cotranscribed from three putative promoters located upstream of cphB1, and, additionally, the cphA1 gene was expressed monocistronically from at least two promoters located in the intergenic cphB1-cphA1 region. Both constitutive promoters and promoters dependent on the global nitrogen control transcriptional regulator NtcA were identified. In cluster cph2, the cphB2 and cphA2 genes, which are found in opposite orientations, were expressed as monocistronic messages in media containing ammonium, nitrate, or N(2), but expression was higher in the absence of ammonium. Expression of the cph2 genes was lower than that of cph1 genes. Analysis of cph gene insertional mutants indicated that cluster cph1 genes contributed more than cluster cph2 genes to cyanophycin accumulation in the whole filament as well as in heterocysts. Diazotrophic growth was more severely impaired in cyanophycinase than in cyanophycin synthetase mutants, indicating that cyanophycin, although normally synthesized in the heterocysts, is not required for heterocyst function and that the inability to degrade this polymer is detrimental for the diazotrophic growth of the cyanobacterium.

Cytochrome c oxidase genes required for nitrogenase activity and diazotrophic growth in Anabaena sp. PCC 7120.

N2 fixation is an O2-sensitive process and some filamentous diazotrophic cyanobacteria that grow performing oxygenic photosynthesis confine their N2 fixation machinery to heterocysts, specialized cells that maintain a reducing environment adequate for N2 fixation. Respiration is thought to contribute to the diazotrophic metabolism of heterocysts and the genome of the heterocyst-forming cyanobacterium Anabaena sp. PCC 7120 bears three gene clusters putatively encoding cytochrome c oxidases. Transcript analysis of these cox gene clusters through RNA/DNA hybridization identified two cox operons, cox2 and cox3, that are induced after nitrogen step-down in an NtcA- and HetR-dependent manner and appear to be expressed specifically in heterocysts. In contrast, cox1 was expressed only in vegetative cells. Expression of cox2 and cox3 occurred at an intermediate stage (about 9 h) during the process of heterocyst development following nitrogen step-down. Inactivation of genes in the two inducible cox operons, but not separately in either of them, strongly reduced nitrogenase activity and prevented diazotrophic growth in aerobic conditions. These results show that the nitrogen-regulated cytochrome c oxidase-type respiratory terminal oxidases Cox2 and Cox3 are essential for heterocyst function in Anabaena sp. PCC 7120.