Heterocyst and akinete induction with altered pattern in Anabaena cylindrica,caused by neo-peptone

Neo-peptone B119 (Difco) was found to have a significant effect on differentiation of heterocysts and akinetes in Anabaena cylindrica. On adding neopeptone (0.4 g/l) to exponential phase culture of A. cylindrica, the following effects were observed (i) increased heterocyst frequency with altered heterocyst spacing and presence of double and multiple heterocysts after 24 h in cultures grown on N-free medium, (ii) induction of regular pattern of heterocysts after 48 h, in culture grown on medium supplemented with NH₄Cl, (iii) induction of pro-akinetes after 48 h in both N-free and ammonium-grown cultures. The higher concentrations of neo-peptone were lytic to A. cylindrica, and, its lytic and inductive effects could be decreased by acid hydrolysis or supplementation of NH₄Cl. Gel-filtration of neo-peptone showed that the inductive as well as the lytic effect was associated with some active factor(s) with molecular weight between 10,000–20,000. The retention of the inductive effect on autoclavation but its loss on trypsin digestion suggested that active factor(s) may be heat stable polypeptide(s). The heterocyst induction by active factor(s) decreased and akinete induction increased with increasing culture age. The pro-akinetes induced during exponential phase divided before maturation, while those induced during late exponential phase, could achieve full maturity. Growth and nitrogenase activity was unaffected while there was an increase in mean cell length on treatment of A. cylindrica with active factor(s) from neo-peptone, indicating that the effect may be mediated through cell division process(es).Abbreviations used N Nitrogen - chl chlorophyll     

Effect of nitrogen starvation on the morphology and ultrastructure of the cyanobacterium Mastigocladus laminosus.

The effects of nitrogen starvation on the morphology and ultrastructure of the branching, filamentous cyanobacterium Mastigocladus laminosus were examined with light and electron microscopy. The internal ultrastructural characteristics of vegetative cells changed markedly during nitrogen starvation. Carboxysomes were degraded, while polyphosphate bodies and lipid bodies accumulated. The ultrastructure of mature heterocysts was also affected by nitrogen starvation; their intracytoplasmic membranes vesiculated to form vacuolelike structures and, eventually, large empty regions in the cytoplasm. Nitrogen starvation stimulated extensive heterocyst differentiation in M. laminosus, producing heterocyst frequencies of 17.5% in narrow filaments and 28.3% in wide filaments within 44 h after transfer to N-free conditions. Cells in wide filaments differentiated so extensively that only 16.8% of them failed to initiate the differentiation process within 44 h.     

Movement of Carbon from Vegetative Cells to Heterocysts in Anabaena cylindrica

Radioactive carbon assimilated by vegetative cells of Anabaena cylindrica in the light passed via an intrafilamentous route into heterocysts in the dark. After several hours, label per heterocyst approximated label per vegetative cell. Much of the label entering heterocysts was not available for diffusional exchange back into vegetative cells.     

Changes in thylakoid structure associated with the differentiation of heterocysts in the cyanobacterium, Anabaena cylindrica.

The thylakoids of vegetative cells of the filamentous cyanobacterium, Anabaena cylindrica, are capable of oxygen-evolving photosynthesis and contain both Photosystems I and II (PSI and PSII). The heterocysts, cells specialized for nitrogen fixation, do not produce oxygen and lack Photosystem II activity, the major accessory pigments, and perhaps the chlorophyll a associated with PSII. Freeze-fracture replicas of vegetative cells and of heterocysts reveal differences in the structure of the thylakoids. A histogram of particle sizes on the exoplasmic fracture face (E-face, EF) of vegetative cell thylakoids has two major peaks, at 75 and 100 A. The corresponding histogram for heterocyst thylakoids lacks the 100 A size class, but has a very large peak at about 55 A with a shoulder at 75 A. Histograms of protoplasmic fracture face (P-face, PF) particle diameters show single broad peaks, the mean diameter being 71 A for vegetative cells and 64 A for heterocysts. The thylakoids of both cell types have about 5600 particles/micrometers2 on the P-face. On the E-face, the density drops from 939 particles/micrometers2 on vegetative cell thylakoids to 715 particles/micrometers2 on heterocyst thylakoids. The data suggest that the 100 A E-face particle of vegetative cell thylakoids is a PSII complex. The 55 A EF particle of heterocysts may be part of the nitrogenase complex or a remnant of the PSII complex. The role of the 75 A EF particle is unknown. Other functions localized on cyanobacterial thylakoids, such as respiration and hydrogenase activity, must be considered when interpreting the structure of these complex thylakoids.     

Nitrogenase in free-living and symbiotic cyanobacteria: Immunoelectron microscopic localization

Abstract Nitrogenase (Fe-protein) was localized in the free-living cyanobacterium Anabaena cylindrica and in the cyanobionts of Cycas revoluta and Peltigera aphthosa , using colloidal gold as an immunocytochemical marker. The Fe-protein was found to be evenly distributed throughout the heterocyst cytoplasma in A. cylindrica and in both the cyanobionts, including multiple heterocysts of the C. revoluta cyanobiont. No label was observed in the vegetative cells of free-living A. cylindrica or of the cyanobionts, although the cyanobionts apparently live under microaerobic conditions.     

HETEROCYST DEVELOPMENT AND LOCALIZATION OF CYANOPHYCIN IN N2-FIXING CULTURES OF ANABAENA SP. PCC 7120 (CYANOBACTERIA)

The process of N₂ fixation in the filamentous cyanobacterium Anabaena sp. PCC 7120 is known to occur in terminally differentiated cells called heterocysts. This study is concerned with a morphological and immunocytochemical analysis of the developing heterocysts. The heterocysts continue a developmental process after synthesis of the specialized cell wall and the formation of the proheterocyst. The initial stages were described by Wilcox et al. (1973) and designated stages 1 through 7, with stages 5–7 associated with the maturing heterocyst. We now designate a stage 8 as the postmaturation stage, based on physiological and ultrastructural evidence. Immunocytochemistry to detect the nitrogenase protein NifH and the nonribosomally synthesized polypeptide cyanophycin demonstrated a complementary accumulation of these polypeptides. Accumulation of the nitrogenase protein was greatest at stages 5 and 6 and then declined precipitously. Cyanophycin was more prevalent after late stage 6 and was primarily associated with the polar nodule (polar plug) and the neck connecting the heterocyst with the adjoining vegetative cell. We suggest that the cyanophycin-containing polar plug is a key intermediate in the storage of fixed nitrogen in the heterocyst, a result consistent with the suggestion first made by Carr (1988) that cyanophycin exists as a dynamic reservoir of fixed nitrogen within the heterocysts.     

Boron Requirement for Envelope Structure and Function in Anabaena PCC 7119 Heterocysts

The effects of boron deficiency on the ultrastructure and envelopecomposition of heterocysts in the filamentous cyanobacteriumAnabaena PCC 7119 were studied. Microscopic examination of boron-deficientcultures showed changes in heterocyst morphology. When thesecells were isolated and their glycolipid content determined,this specific component of the laminated layer of the heterocystenvelope was found to be lacking. The evidence presented supportsthe view that boron plays an essential role in the structureand function of the heterocyst envelope. Key words: Anabaena, boron, heterocysts, nitrogenase, oxygen-protection     

The 2A resolution crystal structure of HetL, a pentapeptide repeat protein involved in regulation of heterocyst differentiation in the cyanobacterium Nostoc sp. strain PCC 7120

The hetL gene from the cyanobacterium Nostoc sp. PCC 7120 encodes a 237 amino acid protein (25.6kDa) containing 40 predicted tandem pentapeptide repeats. Nostoc sp. PCC 7120 is a filamentous cyanobacterium that forms heterocysts, specialized cells capable of fixing atmospheric N(2) during nitrogen starvation in its aqueous environment. Under these conditions, heterocysts occur in a regular pattern of approximately one out of every 10-15 vegetative cells. Heterocyst differentiation is highly regulated involving hundreds of genes, one of which encodes PatS, thought to be an intercellular peptide signal made by developing heterocysts to inhibit heterocyst differentiation in neighboring vegetative cells, thus contributing to pattern formation and spacing of heterocysts along the filament. While overexpression of PatS suppresses heterocyst differentiation in Nostoc sp. PCC 7120, overexpression of HetL produces a multiple contiguous heterocyst phenotype with loss of the wild type heterocyst pattern, and strains containing extra copies of hetL allow heterocyst formation even in cells overexpressing PatS. Thus, HetL appears to interfere with heterocyst differentiation inhibition by PatS, however, the mechanism for HetL function remains unknown. As a first step towards exploring the mechanism for its biochemical function, the crystal structure of HetL has been solved at 2.0A resolution using sulfur anomalous scattering.     

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.     

A TolC-like protein is required for heterocyst development in Anabaena sp. strain PCC 7120

The filamentous cyanobacterium Anabaena sp. strain PCC 7120 forms heterocysts in a semiregular pattern when it is grown on N2 as the sole nitrogen source. The transition from vegetative cells to heterocysts requires marked metabolic and morphological changes. We show that a trimeric pore-forming outer membrane beta-barrel protein belonging to the TolC family, Alr2887, is up-regulated in developing heterocysts and is essential for diazotrophic growth. Mutants defective in Alr2887 did not form the specific glycolipid layer of the heterocyst cell wall, which is necessary to protect nitrogenase from external oxygen. Comparison of the glycolipid contents of wild-type and mutant cells indicated that the protein is not involved in the synthesis of glycolipids but might instead serve as an exporter for the glycolipid moieties or enzymes involved in glycolipid attachment. We propose that Alr2887, together with an ABC transporter like DevBCA, is part of a protein export system essential for assembly of the heterocyst glycolipid layer. We designate the alr2887 gene hgdD (heterocyst glycolipid deposition protein).     

Molecular cloning and characterization of hetR genes from filamentous cyanobacteria

HetR, a serine type protease, plays an important role in heterocyst differentiation in filamentous cyanobacteria. We isolated and sequenced the hetR genes from different heterocystous and filamentous nonheterocystous cyanobacteria. The hetR gene in the heterocyst forming Anabaena variabilis ATCC 29413 FD was interrupted by interposon mutagenesis (mutant strain WSIII8). This mutant does not form heterocysts and shows no diazotrophic growth under aerobic conditions. However, under anaerobic N(2)-fixing conditions, the WSIII8 cells are able to grow, and high nitrogenase (Nif2) activity is detectable. Nif2 expression was demonstrated in each vegetative cell of the filament by immunolocalization 4 h after nitrogen step-down.     

Tracing the path of a prokaryotic paracrine signal

Filamentous heterocyst‐forming cyanobacteria are a beautiful example of prokaryotic multicellularity. The filaments can achieve simultaneous nitrogen fixation and oxygenic photosynthesis by cooperation between two cell types: the photosynthetic vegetative cells and the nitrogen‐fixing heterocysts. The multicellular features exhibited by the system include differentiation of different cell types, metabolic interdependence and even pattern formation, as the spacing of heterocysts along the filament is non‐random. Recent years have seen exciting progress both in understanding the control of heterocyst differentiation, and also in understanding the function of ‘septal junctions’: an array of pore‐like structures at the cell junctions that allow intercellular communication by facilitating the diffusion of small molecules from cell to cell. A new report by Rivers et al. (2014) makes the connection between pattern formation and intercellular communication by showing that a mutation that partially disables the septal junctions leads to a decrease in the range of a signal dependent on the HetN protein that is one of the factors controlling heterocyst spacing. This suggests that the signal travels from cell to cell by diffusion through the septal junctions, opening the door to quantitative understanding of the mechanism that controls heterocyst spacing in filamentous cyanobacteria.     

Cyanobacterial Cell Lineage Analysis of the Spatiotemporal hetR Expression Profile during Heterocyst Pattern Formation in Anabaena sp. PCC 7120

Diazotrophic heterocyst formation in the filamentous cyanobacterium, Anabaena sp. PCC 7120, is one of the simplest pattern formations known to occur in cell differentiation. Most previous studies on heterocyst patterning were based on statistical analysis using cells collected or observed at different times from a liquid culture, which would mask stochastic fluctuations affecting the process of pattern formation dynamics in a single bacterial filament. In order to analyze the spatiotemporal dynamics of heterocyst formation at the single filament level, here we developed a culture system to monitor simultaneously bacterial development, gene expression, and phycobilisome fluorescence. We also developed micro-liquid chamber arrays to analyze multiple Anabaena filaments at the same time. Cell lineage analyses demonstrated that the initial distributions of hetR::gfp and phycobilisome fluorescence signals at nitrogen step-down were not correlated with the resulting distribution of developed heterocysts. Time-lapse observations also revealed a dynamic hetR expression profile at the single-filament level, including transient upregulation accompanying cell division, which did not always lead to heterocyst development. In addition, some cells differentiated into heterocysts without cell division after nitrogen step-down, suggesting that cell division in the mother cells is not an essential requirement for heterocyst differentiation.     

Heterocyst differentiation and pattern formation in cyanobacteria: a chorus of signals

Heterocyst differentiation in filamentous cyanobacteria provides an excellent prokaryotic model for studying multicellular behaviour and pattern formation. In Anabaena sp. strain PCC 7120, for example, 5-10% of the cells along each filament are induced, when deprived of combined nitrogen, to differentiate into heterocysts. Heterocysts are specialized in the fixation of N(2) under oxic conditions and are semi-regularly spaced among vegetative cells. This developmental programme leads to spatial separation of oxygen-sensitive nitrogen fixation (by heterocysts) and oxygen-producing photosynthesis (by vegetative cells). The interdependence between these two cell types ensures filament growth under conditions of combined-nitrogen limitation. Multiple signals have recently been identified as necessary for the initiation of heterocyst differentiation, the formation of the heterocyst pattern and pattern maintenance. The Krebs cycle metabolite 2-oxoglutarate (2-OG) serves as a signal of nitrogen deprivation. Accumulation of a non-metabolizable analogue of 2-OG triggers the complex developmental process of heterocyst differentiation. Once heterocyst development has been initiated, interactions among the various components involved in heterocyst differentiation determine the developmental fate of each cell. The free calcium concentration is crucial to heterocyst differentiation. Lateral diffusion of the PatS peptide or a derivative of it from a developing cell may inhibit the differentiation of neighbouring cells. HetR, a protease showing DNA-binding activity, is crucial to heterocyst differentiation and appears to be the central processor of various early signals involved in the developmental process. How the various signalling pathways are integrated and used to control heterocyst differentiation processes is a challenging question that still remains to be elucidated.     

Heterocyst patterns without patterning proteins in cyanobacterial filaments

We have quantitatively modeled heterocyst differentiation after fixed nitrogen step-down in the filamentous cyanobacterium Anabaena sp. PCC 7120 without lateral inhibition due to the patterning proteins PatS or HetN. We use cell growth and division together with fixed-nitrogen dynamics and allow heterocysts to differentiate upon the local exhaustion of available fixed nitrogen. Slow transport of fixed nitrogen along a shared periplasmic space allows for fast growing cells to differentiate ahead of their neighbors. Cell-to-cell variability in growth rate determines the initial heterocyst pattern. Early release of fixed nitrogen from committed heterocysts allows a significant fraction of vegetative cells to be retained at later times. We recover the experimental heterocyst spacing distributions and cluster size distributions of Khudyakov and Golden [Khudyakov, I.Y., Golden, J.W., 2004. Different functions of HetR, a master regulator of heterocyst differentiation in Anabaena sp PCC 7120, can be separated by mutation. Proc. Natl. Acad. Sci. U. S. A. 101, 16040-16045].     

Anaerobic and aerobic hydrogen gas formation by the blue-green alga Anabaena cylindrica.

An investigation was made of certain factors involved in the formation of hydrogen gas, both in an anaerobic environment (argon) and in air, by the blue-green alga Anabaena cylindrica. The alga had not been previously adapted under hydrogen gas and hence the hydrogen evolution occurred entirely within the nitrogen-fixing heterocyst cells; organisms grown in a fixed nitrogen source, and which were therefore devoid of heterocysts, did not produce hydrogen under these conditions. Use of the inhibitor dichlorophenyl-dimethyl urea showed that hydrogen formation was directly dependent on photosystem I and only indirectly dependent on photosystem II, consistent with heterocysts being the site of hydrogen formation. The uncouplers carbonyl cyanide chlorophenyl hydrazone and dinitrophenol almost completely inhibited hydrogen formation, indicating that the process occurs almost entirely via the adenosine 5'-triphosphate-dependent nitrogenase. Salicylaldoxime also inhibited hydrogen formation, again illustrating the necessity of photophosphorylation. Whereas hydrogen formation could usually only be observed in anaerobic, dinitrogen-free environments, incubation in the presence of the dinitrogen-fixing inhibitor carbon monoxide plus the hydrogenase inhibitor acetylene resulted in significant formation of hydrogen even in air. Hydrogen formation was studied in batch cultures as a function of age of the cultures and also as a function of culture concentration, in both cases the cultures being harvested in logarithmic growth. Hydrogen evolution (and acetylene-reducing activity) exhibited a distinct maximum with respect to the age of the cultures. Finally, the levels of the protective enzyme, superoxide dismutase, were measured in heterocyst and vegetative cell fractions of the organism; the level was twice as high in heterocyst cells (2.3 units/mg of protein) as in vegetative cells (1.1 units/mg of protein). A simple procedure for isolating heterocyst cells is described.     

Anaerobic and Aerobic Hydrogen Gas Formation by the Blue-Green Alga Anabaena cylindrica

An investigation was made of certain factors involved in the formation of hydrogen gas, both in an anaerobic environment (argon) and in air, by the blue-green alga Anabaena cylindrica. The alga had not been previously adapted under hydrogen gas and hence the hydrogen evolution occurred entirely within the nitrogen-fixing heterocyst cells; organisms grown in a fixed nitrogen source, and which were therefore devoid of heterocysts, did not produce hydrogen under these conditions. Use of the inhibitor dichlorophenyl-dimethyl urea showed that hydrogen formation was directly dependent on photosystem I and only indirectly dependent on photosystem II, consistent with heterocysts being the site of hydrogen formation. The uncouplers carbonyl cyanide chlorophenyl hydrazone and dinitrophenol almost completely inhibited hydrogen formation, indicating that the process occurs almost entirely via the adenosine 5′-triphosphate-dependent nitrogenase. Salicylaldoxime also inhibited hydrogen formation, again illustrating the necessity of photophosphorylation. Whereas hydrogen formation could usually only be observed in anaerobic, dinitrogen-free environments, incubation in the presence of the dinitrogen-fixing inhibitor carbon monoxide plus the hydrogenase inhibitor acetylene resulted in significant formation of hydrogen even in air. Hydrogen formation was studied in batch cultures as a function of age of the cultures and also as a function of culture concentration, in both cases the cultures being harvested in logarithmic growth. Hydrogen evolution (and acetylene-reducing activity) exhibited a distinct maximum with respect to the age of the cultures. Finally, the levels of the protective enzyme, superoxide dismutase, were measured in heterocyst and vegetative cell fractions of the organism; the level was twice as high in heterocyst cells (2.3 units/mg of protein) as in vegetative cells (1.1 units/mg of protein). A simple procedure for isolating heterocyst cells is described.     

Dynamics and Cell-Type Specificity of the DNA Double-Strand Break Repair Protein RecN in the Developmental Cyanobacterium Anabaena sp. Strain PCC 7120

DNA replication and repair are two fundamental processes required in life proliferation and cellular defense and some common proteins are involved in both processes. The filamentous cyanobacterium Anabaena sp. strain PCC 7120 is capable of forming heterocysts for N₂ fixation in the absence of a combined-nitrogen source. This developmental process is intimately linked to cell cycle control. In this study, we investigated the localization of the DNA double-strand break repair protein RecN during key cellular events, such as chromosome damaging, cell division, and heterocyst differentiation. Treatment by a drug causing DNA double-strand breaks (DSBs) induced reorganization of the RecN focus preferentially towards the mid-cell position. RecN-GFP was absent in most mature heterocysts. Furthermore, our results showed that HetR, a central player in heterocyst development, was involved in the proper positioning and distribution of RecN-GFP. These results showed the dynamics of RecN in DSB repair and suggested a differential regulation of DNA DSB repair in vegetative cell and heterocysts. The absence of RecN in mature heterocysts is compatible with the terminal nature of these cells.