The cyanobacterial endosymbiont of the unicellular algae <Emphasis Type="Italic">Rhopalodia gibba</Emphasis> shows reductive genome evolution

Background  

Bacteria occur in facultative association and intracellular symbiosis with a diversity of eukaryotic hosts. Recently, we have helped to characterise an intracellular nitrogen fixing bacterium, the so-called spheroid body, located within the diatom Rhopalodia gibba. Spheroid bodies are of cyanobacterial origin and exhibit features that suggest physiological adaptation to their intracellular life style. To investigate the genome modifications that have accompanied the process of endosymbiosis, here we compare gene structure, content and organisation in spheroid body and cyanobacterial genomes.     

Spheroid bodies in rhopalodiacean diatoms were derived from a single endosymbiotic cyanobacterium

Members of the diatom family Rhopalodiaceae possess cyanobacteria-derived intracellular structures called spheroid bodies (SBs) that very likely carry out nitrogen fixation. Due to the shortage of molecular data from SBs and rhopalodiacean diatoms, it remains unclear how SBs were established and spread in rhopalodiacean diatoms. We here amplified the small subunit ribosomal DNA sequences from both host and SB in three rhopalodiacean diatom species, Epithemia turgida, E. sorex, and Rhopalodia gibba. Phylogenetic analyses considering these new sequences clearly indicate that the SBs were acquired by a common ancestor of rhopalodiacean diatoms and have been retained during host speciation.     

Unicellular cyanobacteria with a new mode of life: the lack of photosynthetic oxygen evolution allows nitrogen fixation to proceed

Some unicellular N₂-fixing cyanobacteria have recently been found to lack a functional photosystem II of photosynthesis. Such organisms, provisionally termed UCYN-A, of the oceanic picoplanktion are major contributors to the global marine N-input by N₂-fixation. Since their photosystem II is inactive, they can perform N₂-fixation during the day. UCYN-A organisms cannot be cultivated as yet. Their genomic analysis indicates that they lack genes coding for enzymes of the Calvin cycle, the tricarboxylic acid cycle and for the biosynthesis of several amino acids. The carbon source in the ocean that allows them to thrive in such high abundance has not been identified. Their genomic analysis implies that they metabolize organic carbon by a new mode of life. These unicellular N₂-fixing cyanobacteria of the oceanic picoplankton are evolutionarily related to spheroid bodies present in diatoms of the family Epithemiaceae, such as Rhopalodia gibba. More recently, spheroid bodies were ultimately proven to be related to cyanobacteria and to express nitrogenase. They have been reported to be completely inactive in all photosynthetic reactions despite the presence of thylakoids. Sequence data show that R. gibba and its spheroid bodies are an evolutionarily young symbiosis that might serve as a model system to unravel early events in the evolution of chloroplasts. The cell metabolism of UCYN-A and the spheroid bodies may be related to that of the acetate photoassimilating green alga Chlamydobotrys.     

EFFECTS OF NITROGEN AND PHOSPHORUS ON THE ENDOSYMBIONT LOAD OF RHOPALODIA GIBBA AND EPITHEMIA TURGIDA (BACILLARIOPHYCEAE)1

Diatoms of the family Epithemiaceae possess a unicellular nitrogen-fixing cyanobacterial endosymbiont. We investigated the potential of extracellular nitrogen and phosphorus concentrations to affect the endosymbiont load of Rhopalodia gibba O. Müll, and Epithemia turgida Ehr. in field and culture populations. In a growth chamber experiment, monoclonal cultures of R. gibba were exposed to three levels of nitrate-nitrogen. Nutrient-diffusing substrates were used in a lake environment to create nine microhabitats of varying nitrogen and phosphorus ratios for natural populations of R. gibba and E. turgida. The number of endosymbionts per diatom increased as ambient nitrogen became limiting; mean endosymbiont volume increased as nitrogen increased. The mean endosymbiont surface area: volume ratio decreased with increasing nitrogen. Total endosymbiont volume per diatom (the product of the number of endosymbionts per diatom and their individual biovolumes) did not have a simple response to increasing nitrogen. Phosphorus limitation uncoupled the relationship between endosymbiont load and nitrogen. We suspect that flexibility of the endosymbiont load can reduce the metabolic cost to the diatom if the endosymbionts are dependent on the diatom for a resource.     

Genomes at the interface between bacteria and organelles

The topic of the transition of the genome of a free-living bacterial organism to that of an organelle is addressed by considering three cases. Two of these are relatively clear-cut as involving respectively organisms (cyanobacteria) and organelles (plastids). Cyanobacteria are usually free-living but some are involved in symbioses with a range of eukaryotes in which the cyanobacterial partner contributes photosynthesis, nitrogen fixation, or both of these. In several of these symbioses the cyanobacterium is vertically transmitted, and in a few instances, sufficient unsuccessful attempts have been made to culture the cyanobiont independently for the association to be considered obligate for the cyanobacterium. Plastids clearly had a cyanobacterial ancestor but cannot grow independently of the host eukaryote. Plastid genomes have at most 15% of the number of genes encoded by the cyanobacterium with the smallest number of genes; more genes than are retained in the plastid genome have been transferred to the eukaryote nuclear genome, while the rest of the cyanobacterial genes have been lost. Even the most cyanobacteria-like plastids, for example the "cyanelles" of glaucocystophyte algae, are functionally and genetically very similar to other plastids and give little help in indicating intermediates in the evolution of plastids. The third case considered is the vertically transmitted intracellular bacterial symbionts of insects where the symbiosis is usually obligate for both partners. The number of genes encoded by the genomes of these obligate symbionts is intermediate between that of organelles and that of free-living bacteria, and the genomes of the insect symbionts also show rapid rates of sequence evolution and AT (adenine, thymine) bias. Genetically and functionally, these insect symbionts show considerable similarity to organelles.     

Presequence Acquisition During Secondary Endocytobiosis and thePossible Role of Introns

Targeting of nucleus-encoded proteins into chloroplasts is mediated by N-terminal presequences. During evolution of plastids from formerly free-living cyanobacteria by endocytobiosis, genes for most plastid proteins have been transferred from the plastid genome to the nucleus and subsequently had to be equipped with such plastid targeting sequences. So far it is unclear how the gene domains coding for presequences and the respective mature proteins may have been assembled. While land plant plastids are supposed to originate from a primary endocytobiosis event (a prokaryotic cyanobacterium was taken up by a eukaryotic cell), organisms with secondary plastids like diatoms experienced a second endocytobiosis step involving a eukaryotic alga taken up by a eukaryotic host cell. In this group of algae, apparently most genes encoding chloroplast proteins have been transferred a second time (from the nucleus of the endosymbiont to the nucleus of the secondary host) and thus must have been equipped with additional targeting signals. We have analyzed cDNAs and the respective genomic DNA fragments of seven plastid preproteins from the diatom Phaeodactylum tricornutum. In all of these genes we found single spliceosomal introns, generally located within the region coding for the N-terminal plastid targeting sequences or shortly downstream of it. The positions of the introns can be related to the putative phylogenetic histories of the respective genes, indicating that the bipartite targeting sequences in these secondary algae might have evolved by recombination events via introns.The nucleotide sequences have been deposited at Genbank under accession numbers AY191862, AY191863, AY191864, AY191865, AY191866, AY191867, and AY191868.     

The luggage hypothesis: Comparisons of two phototrophic hosts with nitrogen-fixing cyanobacteria and implications for analogous life strategies for kleptoplastids/secondary symbiosis in dinoflagellates

Nostoc and Richelia belong to a group of heterocystous cyanobacteria and are unique within this group in forming intracellular symbioses with phototrophic hosts, the angiosperm Gunnera and the diatoms (algae) Rhizosolenia and Hemiaulus, respectively. The function of the cyanobiont is similar in the symbioses, namely providing fixed atmospheric nitrogen to their hosts; also the cyanobionts are contained in a host compartment, the symbiosome. The evolutionary timescale for the cyanobiont-endosymbiosis formation is in both instances about ≈90 Ma. However, the potentials for further co-evolution of host and microsymbiont, are different. Nostoc is regarded as preyed upon by its host, while in the Richelia-Rhizosolenia symbiosis example the evolution towards a new type of permanent organelle is possible. It is proposed that symbiosis is ruled by divergent host strategies. In the case of Richelia-Rhizosolenia the evolution of a permanent symbiosis is linked to diatom hosts needing to carry the cyanobiont permanently, as it is not available free-living in the oceans. However, in the case of Nostoc/Gunnera, the host exploits an abundant cyanobacterial species. A model where the relative abundance of microsymbionts determines the nature of the symbiosis comes into view: If environmental ratios of host/microsymbiont are so that hosts are the dominating party, then the host has to carry the microsymbiont as luggage (vertical transmission). Likewise, if the ratio of microsymbiont is higher than host, than the host will prey on the microsymbiont (horizontal transmission). The article also discusses the retention of secondary plastids in dinoflagellates. We show that dinoflagellates are organisms that exemplify both types of strategies that is either preying or harbouring a permanent organelle. The difference from the cyanobacterial example is that only parts of the eukaryotic microsymbionts are kept, usually only the plastid. We emphasize that the dinoflagellates can obtain their plastids from various different organisms. The luggage theory offers an explanation to why some dinoflagellate species contain kleptoplastids, while others have permanent, secondary plastids and some have tertiary plastids.     

Metabolic symbiosis and the birth of the plant kingdom

Eukaryotic cells are composed of a variety of membrane-bound organelles that are thought to derive from symbiotic associations involving bacteria, archaea, or other eukaryotes. In addition to acquiring the plastid, all Archaeplastida and some of their endosymbiotic derivatives can be distinguished from other organisms by the fact that they accumulate starch, a semicrystalline-storage polysaccharide distantly related to glycogen and never found elsewhere. We now provide the first evidence for the existence of starch in a particular species of single-cell diazotrophic cyanobacterium. We provide evidence for the existence in the eukaryotic host cell at the time of primary endosymbiosis of an uridine diphosphoglucose (UDP-glucose)-based pathway similar to that characterized in amoebas. Because of the monophyletic origin of plants, we can define the genetic makeup of the Archaeplastida ancestor with respect to storage polysaccharide metabolism. The most likely enzyme-partitioning scenario between the plastid's ancestor and its eukaryotic host immediately suggests the precise nature of the ancient metabolic symbiotic relationship. The latter consisted in the export of adenosine diphosphoglucose (ADP-glucose) from the cyanobiont in exchange for the import of reduced nitrogen from the host. We further speculate that the monophyletic origin of plastids may lie in an organism with close relatedness to present-day group V cyanobacteria.     

Phylogenomics Uncovers Evolutionary Trajectory of Nitrogen Fixation in Cyanobacteria

Biological nitrogen fixation (BNF) by cyanobacteria is of significant importance for the Earth’s biogeochemical nitrogen cycle but is restricted to a few genera that do not form monophyletic group. To explore the evolutionary trajectory of BNF and investigate the driving forces of its evolution, we analyze 650 cyanobacterial genomes and compile the database of diazotrophic cyanobacteria based on the presence of nitrogen fixation gene clusters (NFGCs). We report that 266 of 650 examined genomes are NFGC-carrying members, and these potentially diazotrophic cyanobacteria are unevenly distributed across the phylogeny of Cyanobacteria, that multiple independent losses shaped the scattered distribution. Among the diazotrophic cyanobacteria, two types of NFGC exist, with one being ancestral and abundant, which have descended from diazotrophic ancestors, and the other being anaerobe-like and sparse, possibly being acquired from anaerobic microbes through horizontal gene transfer. Interestingly, we illustrate that the origin of BNF in Cyanobacteria coincide with two major evolutionary events. One is the origin of multicellularity of cyanobacteria, and the other is concurrent genetic innovations with massive gene gains and expansions, implicating their key roles in triggering the evolutionary transition from nondiazotrophic to diazotrophic cyanobacteria. Additionally, we reveal that genes involved in accelerating respiratory electron transport (coxABC), anoxygenic photosynthetic electron transport (sqr), as well as anaerobic metabolisms (pfor, hemN, nrdG, adhE) are enriched in diazotrophic cyanobacteria, representing adaptive genetic signatures that underpin the diazotrophic lifestyle. Collectively, our study suggests that multicellularity, together with concurrent genetic adaptations contribute to the evolution of diazotrophic cyanobacteria.     

Biological nitrogen fixation in a post-volcanic chronosequence from south-central Chile

Biological nitrogen fixation is a key ecosystem function incorporating new nitrogen (N) during primary successions. Increasing evidence from tropical and northern temperate forests shows that phosphorus (P) and molybdenum (Mo) either alone or in combination limit the activity of free-living diazotrophs. In this study, we evaluated the effects of Mo, P, and carbon (C) addition, either singly or in combination, and moisture, on diazotrophic activity in a post-volcanic forest chronosequence in south-fentral Chile. Diazotrophic activity, both free-living (associated with fine litter) and symbiotic (associated with the moss Racomitrium lanuginosum and the cyanolichens Pseudocyphellaria berberina and P. coriifolia), was evaluated by incubation of samples and subsequent acetylene reduction assays conducted in the field and laboratory, in winter, spring and autumn of two consecutive years. Results showed that diazotrophic activity varied with the season of the year (lowest during the drier spring season), successional stage (highest in the maximal stage), and N-fixer community type (highest in symbiotic diazotrophs). In general, C+P+Mo limitation was documented for heterotrophic diazotrophs and P+Mo limitation for symbiotic diazotrophs. Limitation of diazotrophic activity was not associated with soil nutrient and C status in the chronosequence. Strong inhibition of diazotrophic activity by high N addition and by low moisture suggests that reductions in precipitation expected for south-central Chile under climate change, as well as increasing inputs of reactive N from atmospheric deposition due to increasing use of N fertilizers, may drastically alter the composition and functional role of cryptogamic assemblages in native forests.     

Nucleus-to-nucleus gene transfer and protein retargeting into a remnant cytoplasm of cryptophytes and diatoms

The complex plastid of the cryptophyte Guillardia theta and of the diatom Phaeodactylum tricornutum can both be traced back to an engulfed eukaryotic red alga. The eukaryotic origin of these plastids is most obvious in cryptophytes, where the organelle still possesses a remnant nucleus, the nucleomorph. The nucleomorph itself is embedded in the periplastid compartment (PPC), the remnant of the former red algal cytosol. In the cryptophyte and diatom, the complex plastid is surrounded by 4 membranes, the outer one being continuous with the host rough endoplasmatic reticulum. In a recent report, we have shown that a nuclear encoded PPC protein of G. theta expressed in P. tricornutum leads to a localization, recently described as being a "bloblike structure," which can be obtained by mutation of plastid protein-targeting sequences of the diatom itself. Here we present further nucleus-encoded PPC proteins from G. theta, such as the eukaryotic translation elongation factor-1alpha, evidence for their nucleus-to-nucleus gene transfer, and retargeting of the proteins. We also investigated the first nuclear encoded PPC-targeted protein of P. tricornutum (Hsp70) and analyzed it for in vivo localization together with the identified G. theta PPC proteins. This revealed that all localize to the bloblike structures, which we suggest is the highly reduced PPC of P. tricornutum. Furthermore, the described cryptophyte PPC proteins possibly allow the elucidation of the processes by which proteins are involved in different levels of host control over its eukaryotic organelle.     

白洋淀硅藻分布及其与水环境的关系

记录了白洋淀不同水域发现的硅藻85个种、变种及未定种,隶属于22个科31个属,其中小环藻属(Cyclotella)、菱形藻属(Nitzschia)、美壁藻属(Caloneis)和舟形藻属(Navicula)等为优势类群。在白洋淀东北部景区水域,以梅尼小环藻(Cyclotellameneghiniana)为主;污染严重的入库河道水域,以半裸具席藻(Sellaphora seminulum)和蓝绿舟形藻(Navicula veneta)为代表;村落水域,以菱形藻属、舟形藻属硅藻为主;航道水域,以颗粒沟链藻(Aulacoseira granulata)为代表;荷塘以弯棒杆藻(Rhopalodia gibba)为代表;而鱼池以菱形藻属硅藻为主。典范对应分析(canonical correspondence analysis,CCA)显示水体总磷浓度(total phosphorus,TP)和pH是影响白洋淀硅藻种群分布的重要因素。利用欧洲硅藻数据库对白洋淀TP和pH做了定量重建,TP为0.13-0.25 mg/L,pH为6.92-7.23,此估测值极为接近实测值。     

Application of a nifH microarray to assess the impact of environmental factors on free-living diazotrophs in a glacier forefield

Glacier forefield environments are exposed to extreme and fluctuating climatic and nutritional conditions. The high diversity of free-living diazotrophic communities found in these environments indicates that nitrogen fixers are able to efficiently cope with such conditions. In this study, a nifH microarray was used to monitor changes in diazotrophic populations in the field over a season, in the presence or absence of plants and in 2 glacier forefields characterized by a different bedrock type (siliceous or calcareous), as well as at different temperatures (10 °C, 15 °C) and under different nitrogen fertilization regimes (0, 10, 40 kg N·ha(-1)·year(-1)) in laboratory systems. Population structures responded highly dynamically to environmental changes. Plant presence had the strongest impact, which decreased toward the end of the season and with high amounts of nitrogen fertilization. Temperature and nitrogen fertilization increases indirectly affected diazotrophic communities through their positive impact on plant growth. These results indicate strong carbon limitation in young glacier forefield soils. Phylotypes related to the genus Methylocystis strongly responded to environmental variations. These methanotrophic microorganisms, which are able to retrieve nitrogen and carbon from the atmospheric pool, are particularly adapted to the extreme nutritional conditions found in glacier forefields.     

Phylogeography of the Microcoleus vaginatus (Cyanobacteria) from three continents--a spatial and temporal characterization

It has long been assumed that cyanobacteria have, as with other free-living microorganisms, a ubiquitous occurrence. Neither the geographical dispersal barriers nor allopatric speciation has been taken into account. We endeavoured to examine the spatial and temporal patterns of global distribution within populations of the cyanobacterium Microcoleus vaginatus, originated from three continents, and to evaluate the role of dispersal barriers in the evolution of free-living cyanobacteria. Complex phylogeographical approach was applied to assess the dispersal and evolutionary patterns in the cyanobacterium Microcoleus vaginatus (Oscillatoriales). We compared the 16S rRNA and 16S-23S ITS sequences of strains which had originated from three continents (North America, Europe, and Asia). The spatial distribution was investigated using a phylogenetic tree, network, as well as principal coordinate analysis (PCoA). A temporal characterization was inferred using molecular clocks, calibrated from fossil DNA. Data analysis revealed broad genetic diversity within M. vaginatus. Based on the phylogenetic tree, network, and PCoA analysis, the strains isolated in Europe were spatially separated from those which originated from Asia and North America. A chronogram showed a temporal limitation of dispersal barriers on the continental scale. Dispersal barriers and allopatric speciation had an important role in the evolution of M. vaginatus. However, these dispersal barriers did not have a permanent character; therefore, the genetic flow among populations on a continental scale was only temporarily present. Furthermore, M. vaginatus is a recently evolved species, which has been going through substantial evolutionary changes.     

Unusual radioresistance of nitrogen-fixing cultures of <Emphasis Type="Italic">Anabaena</Emphasis> strains

Nitrogen-fixing cultures of two species of the filamentous, heterocystous cyanobacterium Anabaena, namely Anabaena sp. strain L-31 and Anabaena torulosa were found to be highly tolerant to ⁶⁰Co gamma radiation. No adverse effect on diazotrophic growth and metabolism were observed up to a dose of 5 kGy. At higher doses, radiation tolerance showed a correspondence with the inherent osmotolerance, with Anabaena L-31 being the more radiation tolerant as well as osmotolerant strain. In Anabaena L-31, exposure to 6 kGy of gamma rays resulted in genome disintegration, but did not reduce viability. Irradiation delayed heterocyst differentiation and nitrogen fixation, and marginally affected diazotrophic growth. All the affected parameters recovered after a short lag, without any discernible postirradiation phenotype. The radiation tolerance of these Gram-negative photoautodiazotrophs is comparable with that of the adiazotrophic photoautotrophic cyanobacterium Chroococcidiopsis or adiazotrophic heterotroph Deinococcus radiodurans. This is the first report of extreme radioresistance in nitrogen-fixing Anabaena cultures.     

Evaluation of nitrogen fixation in the diatom genus Rhizosolenia Ehr. in the absence of its cyanobacterial symbiont Richelia intracellularis Schmidt

Aggregates of two species of Rhizosolenia (R. castracanei andR. imbricata var. shrubsolei) have been reported to containendosymbiotic bacteria and to fix nitrogen. The general importanceof this process to the genus is not known, although of greatpotential significance. Clonal cultures of five species of Rhizosolenia(R. alata, R. bergonii, R. calcar-avis, R. imbricata var. shrubsoleiand R. setigera) were examined for nitrogen fixation using acetylenereduction and compared to cyanobacterial controls. Mecososmaggregates of R. imbricata var. shrubsolei were tested, as wellas chains of R. debyana hand-collected using SCUBA. The Rhizosoleniaspecies did not contain the endosymbiotic cyanobacterium Richeliaintracellularis and were not examined for the presence of endosymbioticbacteria. Nitrogen fixation was not found in the Rhizosoleniaspp.; in contrast, the diazotrophic cyanobacterial controlsOscillatoria erythraea and Anabaena sp. reduced acetylene atsignificant rates. The absence of nitrogen fixation in the Rhizosoleniaspp. suggests diazotrophy by endosymbiotic bacteria is not widespreadin the genus Rhizosolenia, nor is it necessary for survivalof these large diatom cells in nutrient-poor, near-shore waters.     

Concept,Definition, Prevalence,and Host-Interactions of Xenosomes (Cytoplasmic and Nuclear Endosymbionts)1,2

ABSTRACT. In this paper the concept of “xenosome” is greatly expanded from its current usage, which has been based on its application during the past 10 years by Soldo and co-workers solely to certain bacterial invaders of the cytoplasm in species of a single genus of marine scuticociliales. The author proposes that the term now be considered to embrace all DNA-containing, membrane-bounded bodies or organelles—prokaryotic or eukaryotic in original nature—found within the cytoplasm or nucleus of eukaryotic cells of any or all kinds, whether the occupation (“colonization”) is temporary and transient or permanent and stable. Thus, virulent or pathogenically infectious organisms can be included as well as the commonly recognized cell endosymbionts sensu stricto, which are often mutualistic in nature. Of significance, such “normal” cell organelles as plastids, mitochondria, and even nuclei may also be embraced by this expanded definition of xenosome, based on the conjecture that these inclusions might have been “alien” or “foreign” extracellular, independent, free-living organisms in their own past evolutionary histories. The author's enlarged concept and unifying principle allows more meaningful comparative consideration of the numerous and diverse kinds of xenosome-host interrelationships, many of which involve species of protozoa and algae from a large number of the taxonomic groups comprising the kingdom Protista.     

Sub-Cellular Element Analysis of a Cyanobacterium (Nostoc sp.) in Symbiosis with Gunnera manicata by ESI and EELS

Element analysis using electron spectroscopic imaging (ESI) and electron energy loss spectroscopy (EELS) was performed in a symbiotic Nostoc sp. strain found in the upper stem tissue of Gunnera manicata, and in Nostoc PCC 9229, a free-living heterocyst-forming cyanobacterium able to enter into symbiosis with the angiosperm Gunnera in reconstitution experiments. ESI and EELS unequivocally identified the four elements nitrogen (N), sulphur (S), phosphorus (P) and oxygen (O) in different inclusion bodies of these biological specimens. High amounts of nitrogen were solely detected in huge cyanophycin granules in vegetative cells of the symbiotic Nostoc strain, whereas large polyphosphate bodies, containing high amounts of phosphorus, sulphur and oxygen, could be seen in the free-living Nostoc PCC 9229. The latter were usually not present or, when found, very small in vegetative cells of the cyanobiont.     

Cytochrome c6 is the main respiratory and photosynthetic soluble electron donor in heterocysts of the cyanobacterium Anabaena sp. PCC 7120

Cytochrome c₆ is a soluble electron carrier, present in all known cyanobacteria, that has been replaced by plastocyanin in plants. Despite their high structural differences, both proteins have been reported to be isofunctional in cyanobacteria and green algae, acting as alternative electron carriers from the cytochrome b₆-f complex to photosystem I or terminal oxidases. We have investigated the subcellular localization of both cytochrome c₆ and plastocyanin in the heterocyst-forming cyanobacterium Anabaena sp. PCC 7120 grown in the presence of combined nitrogen and under diazotrophic conditions. Our studies conclude that cytochrome c₆ is expressed at significant levels in heterocysts, even in the presence of copper, condition in which it is strongly repressed in vegetative cells. However, the copper-dependent regulation of plastocyanin is not altered in heterocysts. In addition, in heterocysts, cytochrome c₆ has shown to be the main soluble electron carrier to cytochrome c oxidase-2 in respiration. A cytochrome c₆ deletion mutant is unable to grow under diazotrophic conditions in the presence of copper, suggesting that cytochrome c₆ plays an essential role in the physiology of heterocysts that cannot be covered by plastocyanin.