Regulation of Cellular Differentiation in Filamentous Cyanobacteria in Free-Living and Plant-Associated Symbiotic Growth States

Certain filamentous nitrogen-fixing cyanobacteria generate signals that direct their own multicellular development. They also respond to signals from plants that initiate or modulate differentiation, leading to the establishment of a symbiotic association. An objective of this review is to describe the mechanisms by which free-living cyanobacteria regulate their development and then to consider how plants may exploit cyanobacterial physiology to achieve stable symbioses. Cyanobacteria that are capable of forming plant symbioses can differentiate into motile filaments called hormogonia and into specialized nitrogen-fixing cells called heterocysts. Plant signals exert both positive and negative regulatory control on hormogonium differentiation. Heterocyst differentiation is a highly regulated process, resulting in a regularly spaced pattern of heterocysts in the filament. The evidence is most consistent with the pattern arising in two stages. First, nitrogen limitation triggers a nonrandomly spaced cluster of cells (perhaps at a critical stage of their cell cycle) to initiate differentiation. Interactions between an inhibitory peptide exported by the differentiating cells and an activator protein within them causes one cell within each cluster to fully differentiate, yielding a single mature heterocyst. In symbiosis with plants, heterocyst frequencies are increased 3- to 10-fold because, we propose, either differentation is initiated at an increased number of sites or resolution of differentiating clusters is incomplete. The physiology of symbiotically associated cyanobacteria raises the prospect that heterocyst differentiation proceeds independently of the nitrogen status of a cell and depends instead on signals produced by the plant partner.     

Regulation of cellular differentiation in filamentous cyanobacteria in free-living and plant-associated symbiotic growth states.

Certain filamentous nitrogen-fixing cyanobacteria generate signals that direct their own multicellular development. They also respond to signals from plants that initiate or modulate differentiation, leading to the establishment of a symbiotic association. An objective of this review is to describe the mechanisms by which free-living cyanobacteria regulate their development and then to consider how plants may exploit cyanobacterial physiology to achieve stable symbioses. Cyanobacteria that are capable of forming plant symbioses can differentiate into motile filaments called hormogonia and into specialized nitrogen-fixing cells called heterocysts. Plant signals exert both positive and negative regulatory control on hormogonium differentiation. Heterocyst differentiation is a highly regulated process, resulting in a regularly spaced pattern of heterocysts in the filament. The evidence is most consistent with the pattern arising in two stages. First, nitrogen limitation triggers a nonrandomly spaced cluster of cells (perhaps at a critical stage of their cell cycle) to initiate differentiation. Interactions between an inhibitory peptide exported by the differentiating cells and an activator protein within them causes one cell within each cluster to fully differentiate, yielding a single mature heterocyst. In symbiosis with plants, heterocyst frequencies are increased 3- to 10-fold because, we propose, either differentiation is initiated at an increased number of sites or resolution of differentiating clusters is incomplete. The physiology of symbiotically associated cyanobacteria raises the prospect that heterocyst differentiation proceeds independently of the nitrogen status of a cell and depends instead on signals produced by the plant partner.     

Heterocyst differentiation in the cyanobacterium Mastigocladus laminosus.

The morphological and ultrastructural aspects of heterocyst differentiation in the branching, filamentous cyanobacterium Mastigocladus laminosus were examined with light and electron microscopy. The earliest differentiation stages involved cytoplasmic changes, including (i) rapid degradation of carboxysomes, (ii) degradation of polysaccharide granules, and (iii) accumulation of electron-dense ribosomal or protein material (or both). Intermediate differentiation stages involved synthesis of a homogeneous extra wall layer, development of necks leading to adjacent cells, and elaboration of a complex system of intracytoplasmic membranes. Late differentiation stages included further development of necks and continued elaboration of membranes. Mature heterocysts possessed a uniformly electron-dense cytoplasm that contained large numbers of closely packed membranes, some of which were arranged in lamellar stacks. Mature heterocysts lacked all of the inclusion bodies present in undifferentiated vegetative cells, but contained a number of unusual spherical inclusions of variable electron density. Cells in both narrow and wide filaments were capable of differentiating. No regular heterocyst spacing pattern was observed in the narrow filaments; the number of vegetative cells between consecutive heterocysts of any given filament varied by a factor of 10. Heterocysts developed at a variety of locations in the wide, branching filaments, although the majority of them were situated adjacent to branch points. M. laminosus displayed a marked tendency to produce sets of adjacent heterocysts or proheterocysts (or both) that were not separated from each other by vegetative cells. Groups of four or more adjacent heterocysts or proheterocysts occurred frequently in wide filaments, and in some of these filaments virtually all of the cells appeared to be capable of differentiating into heterocysts.     

Isolation of cyanobacterial heterocysts with high and sustained dinitrogen-fixation capacity supported by endogenous reductants

A method is described for the preparation of cyanobacterial heterocysts with high nitrogen-fixation (acetylene-reduction) activity supported by endogenous reductants. The starting material was Anabaena variabilis ATCC 29413 grown in the light in the presence of fructose. Heterocysts produced from such cyanobacteria were more active than those from photoautotrophically-grown A. variabilis, presumably because higher reserves of carbohydrate were stored within the heterocysts. It proved important to avoid subjecting the cyanobacteria to low temperatures under aerobic conditions, as inhibition of respiration appeared to lead to inactivation of nitrogenase. Low temperatures were not harmful in the absence of O₂. A number of potential osmoregulators at various concentrations were tested for use in heterocyst isolation. The optimal concentration (0.2M sucrose) proved to be a compromise between adequate osmotic protection for isolated heterocysts and avoidance of inhibition of nitrogenase by high osmotic strength. Isolated heterocysts without added reductants such as H₂ had about half the nitrogen-fixation activity expected on the basis of intact filaments. H₂ did not increase the rate of acetylene reduction, suggesting that the supply of reductant from heterocyst metabolism did not limit nitrogen fixation under these conditions. Such heterocysts had linear rates of acetylene reduction for at least 2 h, and retained their full potential for at least 12 h when stored at 0°C under N₂.     

Subcellular Localization and Clues for the Function of the HetN Factor Influencing Heterocyst Distribution in Anabaena sp. Strain PCC 7120

In the filamentous cyanobacterium Anabaena sp. strain PCC 7120, heterocysts are formed in the absence of combined nitrogen, following a specific distribution pattern along the filament. The PatS and HetN factors contribute to the heterocyst pattern by inhibiting the formation of consecutive heterocysts. Thus, inactivation of any of these factors produces the multiple contiguous heterocyst (Mch) phenotype. Upon N stepdown, a HetN protein with its C terminus fused to a superfolder version of green fluorescent protein (sf-GFP) or to GFP-mut2 was observed, localized first throughout the whole area of differentiating cells and later specifically on the peripheries and in the polar regions of mature heterocysts, coinciding with the location of the thylakoids. Polar localization required an N-terminal stretch comprising residues 2 to 27 that may represent an unconventional signal peptide. Anabaena strains expressing a version of HetN lacking this fragment from a mutant gene placed at the native hetN locus exhibited a mild Mch phenotype. In agreement with previous results, deletion of an internal ERGSGR sequence, which is identical to the C-terminal sequence of PatS, also led to the Mch phenotype. The subcellular localization in heterocysts of fluorescence resulting from the fusion of GFP to the C terminus of HetN suggests that a full HetN protein is present in these cells. Furthermore, the full HetN protein is more conserved among cyanobacteria than the internal ERGSGR sequence. These observations suggest that HetN anchored to thylakoid membranes in heterocysts may serve a function besides that of generating a regulatory (ERGSGR) peptide.     

14C-labeled metabolites in heterocysts and vegetative cells of Anabaena cylindrica filaments and their presumptive function as transport vehicles of organic carbon and nitrogen

To investigate the transport of primary metabolites in Anabaena cylindrica from vegetative cells into heterocysts, intact filaments were labeled with the heterocysts were separated from the vegetative cells after different time intervals, and the labeling patterns were determined. After a 20-s fixation time, a high percentage of labeling of alanine, glutamate and glutamine, and, to a lesser extent, glucose 6-phosphate was found in heterocysts as compared with whole filaments. The results can be explained if transport of alanine, glutamate, and sugars from vegetative cells into heterocysts is assumed. Alanine can serve as a precursor for reducing equivalents if it is oxidized to glutamine which flows back to the vegetative cells. This idea is supported by an experiment in which exogenous alanine is readily converted by isolated heterocysts to glutamate and glutamine under a N2-H2 atmosphere. The incorporation of [14C]carbonate in isolated heterocysts demonstrated the absence of the reductive pentose phosphate pathway; however, it revealed marked activity of an acid fixation reaction.     

Inactivation of patS and hetN causes lethal levels of heterocyst differentiation in the filamentous cyanobacterium Anabaena sp. PCC 7120

Summary In the filamentous cyanobacterium Anabaena sp. PCC 7120 patS and hetN suppress the differentiation of vegetative cells into nitrogen-fixing heterocysts to establish and maintain a pattern of single heterocysts separated by approximately 10 undifferentiated vegetative cells. Here we show that the patS- and hetN-dependent suppression pathways are the only major factors that prevent vegetative cells from differentiating into heterocysts when a source of ammonia is not present. The patS and hetN pathways are independent of each other, and inactivation of both patS and hetN leads to differentiation of almost all cells of a filament in the absence of a source of fixed nitrogen, compared with approximately 9% in the wild type. Complete differentiation of filaments also occurs when nitrate is supplied as a source of fixed nitrogen, conditions that do not induce differentiation of wild-type filaments. However, ammonia is still capable of suppressing differentiation. The percentage of cells that differentiate into heterocysts appears to be a function of time when a source of fixed nitrogen is absent or a function of growth phase when nitrate is supplied. Although differentiation proceeds unchecked in the absence of patS and hetN expression, differentiation is asynchronous and non-random.     

The isocitrate dehydrogenase from cyanobacteria

The present communication describes the properties of isocitrate dehydrogenase in crude extracts from the unicellular Anacystis nidulans and from heterocysts and vegetative cells of Nostoc muscorum and Anabaena cylindrica. The activity levels of this enzyme are much higher in heterocysts than in vegetative cells of N. muscorum and A. cylindrica. Isocitrate dehydrogenase is virtually inactive in vegetative cells of A. cylindrica. The enzyme is negatively regulated by the reduction charge and scarcely affected by oxoglutarate in the three cyanobacteria. The inhibition by ATP and ADP is competitive with respect to isocitrate and NADP+ in A. cylindrica and N. muscorum and noncompetitive in A. nidulans. Isocitrate dehydrogenase from the three cyanobacteria seems to be a hysteretic enzyme. All the experimental data suggest that the major physiological role of isocitrate and the isocitrate dehydrogenase in heterocysts is not to generate reducing equivalents for N2-fixation. Oxoglutarate formed by the enzyme reaction is likely required for the biosynthesis of glutamate inside the heterocysts. Thioredoxin preparations from spinach chloroplasts or from A. cylindrica activate isocitrate dehydrogenase from either heterocysts or vegetative cells of A. cylindrica. Activation is completed within seconds and requires dithiothreitol besides thioredoxin. The thioredoxin preparation which activates isocitrate dehydrogenase also activates NADP+-dependent malate dehydrogenase from spinach chloroplasts or heterocysts of A. cylindrica. Isocitrate dehydrogenase from A. cylindrica is deactivated by oxidized glutathione. It is speculated that isocitrate dehydrogenase and thioredoxin play a role in the differentiation of vegetative cells to heterocysts.     

Nitrogen fixation by Anabaena cylindrica

Summary Blending Anabaena cylindrica cultures results in a loss of nitrogenase activity which is correlated with the breakage of the filaments at the junctions between heterocysts and vegetative cells. Oxygen inhibition of nitrogen fixation was significant only above atmospheric concentrations. Nitrogen-fixation activities in the dark were up to 50% of those observed in the light and were dependent on oxygen (10 to 20% was optimal). Nitrogenase activity was lost in about 3 h when cells were incubated aerobically in the dark. Re-exposure to light resulted in recovery of nitrogenase activity within 2 h. Blending, oxygen, or dark pre-incubation had similar effects upon cultures grown under air or nitrogen and did not inhibit light-dependent CO₂ fixation. We conclude that heterocysts are the sites of nitrogenase activity and propose a model for nitrogen fixation by Anabaena cylindrica.     

Regulation of photosynthesis during heterocyst differentiation in Anabaena sp. strain PCC 7120 investigated in vivo at single-cell level by chlorophyll fluorescence kinetic microscopy

Changes of photosynthetic activity in vivo of individual heterocysts and vegetative cells in the diazotrophic cyanobacterium Anabaena sp. strain PCC 7120 during the course of diazotrophic acclimation were determined using fluorescence kinetic microscopy (FKM). Distinct phases of stress and acclimation following nitrogen step-down were observed. The first was a period of perception, in which the cells used their internally stored nitrogen without detectable loss of PS II activity or pigments. In the second, the stress phase of nitrogen limitation, the cell differentiation occurred and an abrupt decline of fluorescence yield was observed. This decline in fluorescence was not paralleled by a corresponding decline in photosynthetic pigment content and PS II activity. Both maximal quantum yield and sustained electron flow were not altered in vegetative cells, only in the forming heterocysts. The third, acclimation phase started first in the differentiating heterocysts with a recovery of PS II photochemical yields $F_{\text{v}} /F_{\text{m}} ,\;F^{\prime}_{\text{v}} /F^{\prime}_{\text{m}}.$ F v / F m , F v ′ / F m ′ . Afterwards, the onset of nitrogenase activity was observed, followed by the restoration of antenna pigments in the vegetative cells, but not in the heterocysts. Surprisingly, mature heterocysts were found to have an intact PS II as judged by photochemical yields, but a strongly reduced PS II-associated antenna as judged by decreased F ₀. The possible importance of the functional PS II in heterocysts is discussed. Also, the FKM approach allowed to follow in vivo and evaluate the heterogeneity in photosynthetic performance among individual vegetative cells as well as heterocysts in the course of diazotrophic acclimation. Some cells along the filament (so-called “superbright cells”) were observed to display transiently increased fluorescence yield, which apparently proceeded by apoptosis.     

Formation of glutamine from [13n]ammonia, [13n]dinitrogen, and [14C]glutamate by heterocysts isolated from Anabaena cylindrica.

A method is described for the isolation of metabolically active heterocysts from Anabaena cylindrica. These isolated heterocysts accounted for up to 34% of the acetylene-reducing activity of whole filaments and had a specific activity of up to 1,560 nmol of C2H4 formed per mg of heterocyst chlorphyll per min. Activity of glutamine synthetase was coupled to activity of nitrogenase in isolated heterocysts as shown by acetylene-inhibitable formation of [13N]NH3 and of amidelabeled [13N]glutamine form [13N]N2. A method is also described for the production of 6-mCi amounts of [13N]NH3. Isolated heterocysts formed [13N]glutamine from [13N]NH3 and glutamate, and [14C]glutamine from NH3 and [14C]glutamate, in the presence of magnesium adenosine 5'-triphosphate. Methionine sulfoximine strongly inhibited these syntheses. Glutamate synthase is, after nitrogenase and glutamine synthetase, the third sequential enzyme involved in the assimilation of N2 by intact filaments. However, the kinetics of solubilization of the activity of glutamate synthase during cavitation of suspensions of A. cylindrica indicated that very little, if any, of the activity of that enzyme was located in heterocysts. Concordantly, isolated heterocysts failed to form substantial amounts of radioactive glutamate from either [13N]glutamine or alph-[14C]ketoglutarate in the presence of other substrates and cofactors of the glutamate synthase reaction. However, they formed [14C]glutamate rapidly from alpha-[14C]ketoglutarate by aminotransferase reactions, with various amino acids as the nitrogen donor. The implication of these findings with regard to the identities of the substances moving between heterocysts and vegetative cells are discussed.     

Ultrastructural studies of heterocyst induction by neo-peptone in Anabaena cylindrica

Abstract An ultrastructural study has been performed to elucidate the effect of active polypeptide(s) from neo-peptone on heterocyst induction in Anabaena cylindrica [1]. There was an immediate aggregation of A. cylindrica cells and a clumping of filamentous appendages in the mucilaginous sheath on the addition of active polypeptide(s) from neo-peptone. However, there was no change in the cell wall and cell membrane ultrastructure. An increase in cell length, contortion and disintegration of thylakoids, disappearance of polyphosphate bodies and an accumulation of polyglucose bodies were observed after 18 h of treatment. The double heterocysts induced show a normal heterocyst ultrastructure with well-developed polar nodules between the heterocysts and the vegetative cells, as well as between two heterocysts.
It appears that the inductive effect of active polypeptide(s) from neo-peptone is mediated through their specific binding to filamentous appendages in the mucilaginous sheath.     

Cell Wall Amidase AmiC1 Is Required for Cellular Communication and Heterocyst Development in the Cyanobacterium Anabaena PCC 7120 but Not for Filament Integrity

Filamentous cyanobacteria of the order Nostocales display typical properties of multicellular organisms. In response to nitrogen starvation, some vegetative cells differentiate into heterocysts, where fixation of N(2) takes place. Heterocysts provide a micro-oxic compartment to protect nitrogenase from the oxygen produced by the vegetative cells. Differentiation involves fundamental remodeling of the Gram-negative cell wall by deposition of a thick envelope and by formation of a neck-like structure at the contact site to the vegetative cells. Cell wall-hydrolyzing enzymes, like cell wall amidases, are involved in peptidoglycan maturation and turnover in unicellular bacteria. Recently, we showed that mutation of the amidase homologue amiC2 gene in Nostoc punctiforme ATCC 29133 distorts filament morphology and function. Here, we present the functional characterization of two amiC paralogues from Anabaena sp. strain PCC 7120. The amiC1 (alr0092) mutant was not able to differentiate heterocysts or to grow diazotrophically, whereas the amiC2 (alr0093) mutant did not show an altered phenotype under standard growth conditions. In agreement, fluorescence recovery after photobleaching (FRAP) studies showed a lack of cell-cell communication only in the AmiC1 mutant. Green fluorescent protein (GFP)-tagged AmiC1 was able to complement the mutant phenotype to wild-type properties. The protein localized in the septal regions of newly dividing cells and at the neck region of differentiating heterocysts. Upon nitrogen step-down, no mature heterocysts were developed in spite of ongoing heterocyst-specific gene expression. These results show the dependence of heterocyst development on amidase function and highlight a pivotal but so far underestimated cellular process, the remodeling of peptidoglycan, for the biology of filamentous cyanobacteria.     

Tetrazolium reduction and nitrogenase activity in heterocystous blue-green algae

Summary Heterocysts reduce triphenyl tetrazolium chloride (TTC) faster than vegetative cells apparently because the absence of the O₂-evolving photosystem II and the high electron transport activity in these cells. Although the rate of TTC reduction in vegetative cells is increased by the continuous removal of O₂ evolved in photosynthesis, it has not been possible to obtain rates of TTC reduction comparable with those in heterocysts probably because of the continued competition for electrons between TTC and O₂. The use of nitro-blue tetrazolium chloride (NBT) as a redox indicator has revealed the presence in filaments under aerobic conditions of a gradient of electron transport activity with strongest reducing power in the heterocysts, proheterocysts and vegetative cells next to heterocysts, and with gradually diminishing activity midway between two heterocysts. This pattern is indistinct in filaments grown under micro-aerophilic conditions. The strong electron transport activity in vegetative cells adjacent to heterocysts appears to promote reducing conditions in the heterocysts. Both, red-formazan formation in the heterocysts and blue-formazan deposition in vegetative cells greatly inhibit nitrogenase activity, and this was adversely affected also by the detachment of heterocysts from vegetative cells. The findings are consistent with the idea that the association of heterocysts with vegetative cells in essential for nitrogen fixation to occur in heterocystous blue-green algae.     

Continuous periplasm in a filamentous, heterocyst-forming cyanobacterium

The cyanobacteria bear a Gram-negative type of cell wall that includes a peptidoglycan layer and an outer membrane outside of the cytoplasmic membrane. In filamentous cyanobacteria, the outer membrane appears to be continuous along the filament of cells. In the heterocyst-forming cyanobacteria, two cell types contribute specialized functions for growth: vegetative cells provide reduced carbon to heterocysts, which provide N2-derived fixed nitrogen to vegetative cells. The promoter of the patS gene, which is active specifically in developing proheterocysts and heterocysts of Anabaena sp. PCC 7120, was used to direct the expression of altered versions of the gfp gene. An engineered green fluorescent protein (GFP) that was exported to the periplasm of the proheterocysts through the twin-arginine translocation system was observed also in the periphery of neighbouring vegetative cells. However, if the GFP was anchored to the cytoplasmic membrane, it was observed in the periphery of the producing proheterocysts or heterocysts but not in adjacent vegetative cells. These results show that there is no cytoplasmic membrane continuity between heterocysts and vegetative cells and that the GFP protein can move along the filament in the periplasm, which is functionally continuous and so provides a conduit that can be used for chemical communication between cells.     

The developmental regulator PatD modulates assembly of the cell-division protein FtsZ in the cyanobacterium Anabaena sp. PCC 7120

FtsZ is a tubulin-like GTPase that polymerizes to initiate the process of cell division in bacteria. Heterocysts are terminally differentiated cells of filamentous cyanobacteria that have lost the capacity for cell division and in which the ftsZ gene is downregulated. However, mechanisms of FtsZ regulation during heterocyst differentiation have been scarcely investigated. The patD gene is NtcA dependent and involved in the optimization of heterocyst frequency in Anabaena sp. PCC 7120. Here, we report that the inactivation of patD caused the formation of multiple FtsZ-rings in vegetative cells, cell enlargement, and the retention of peptidoglycan synthesis activity in heterocysts, whereas its ectopic expression resulted in aberrant FtsZ polymerization and cell division. PatD interacted with FtsZ, increased FtsZ precipitation in sedimentation assays, and promoted the formation of thick straight FtsZ bundles that differ from the toroidal aggregates formed by FtsZ alone. These results suggest that in the differentiating heterocysts, PatD interferes with the assembly of FtsZ. We propose that in Anabaena FtsZ is a bifunctional protein involved in both vegetative cell division and regulation of heterocyst differentiation. In the differentiating cells PatD-FtsZ interactions appear to set an FtsZ activity that is insufficient for cell division but optimal to foster differentiation.