Septum-localized protein required for filament integrity and diazotrophy in the heterocyst-forming cyanobacterium Anabaena sp. strain PCC 7120

Heterocysts, formed when filamentous cyanobacteria, such as Anabaena sp. strain PCC 7120, are grown in the absence of combined nitrogen, are cells that are specialized in fixing atmospheric nitrogen (N(2)) under oxic conditions and that transfer fixed nitrogen to the vegetative cells of the filament. Anabaena sp. mutants whose sepJ gene (open reading frame alr2338 of the Anabaena sp. genome) was affected showed filament fragmentation and arrested heterocyst differentiation at an early stage. In a sepJ insertional mutant, a layer similar to a heterocyst polysaccharide layer was formed, but the heterocyst-specific glycolipids were not synthesized. The sepJ mutant did not exhibit nitrogenase activity even when assayed under anoxic conditions. In contrast to proheterocysts produced in the wild type, those produced in the sepJ mutant still divided. SepJ is a multidomain protein whose N-terminal region is predicted to be periplasmic and whose C-terminal domain resembles an export permease. Using a green fluorescent protein translationally fused to the carboxyl terminus of SepJ, we observed that in mature heterocysts and vegetative cells, the protein is localized at the intercellular septa, and when cell division starts, it is localized in a ring whose position is similar to that of a Z ring. SepJ is a novel composite protein needed for filament integrity, proper heterocyst development, and diazotrophic growth.     

Anabaena sp. strain PCC 7120 gene devH is required for synthesis of the heterocyst glycolipid layer

In response to deprivation for fixed nitrogen, the filamentous cyanobacterium Anabaena sp. strain PCC 7120 provides a microoxic intracellular environment for nitrogen fixation through the differentiation of semiregularly spaced vegetative cells into specialized cells called heterocysts. The devH gene is induced during heterocyst development and encodes a product with characteristics of a trans-acting regulatory protein. A devH mutant forms morphologically distinguishable heterocysts but is Fox(-), incapable of nitrogen fixation in the presence of oxygen. We demonstrate that rearrangements of nitrogen fixation genes take place normally in the devH mutant and that it is Fix(+), i.e., has nitrogenase activity under anoxic conditions. The Fox(-) phenotype was shown by ultrastructural studies to be associated with the absence of the glycolipid layer of the heterocyst envelope. The expression of glycolipid biosynthetic genes in the mutant is greatly reduced, and heterocyst glycolipids are undetectable.     

Isolation and preliminary characterization of auxotrophs of a filamentous Cyanobacterium.

Auxotrophic mutants of the filamentous cyanobacterium Anabaena variabilis were isolated by a method in which, after mutagenesis and before penicllin enrichment, mutant and wild-type cells were separated by cavitation. Auxotrophs were identified by their inability to grow on minimal medium, and they were partially characterized by replica plating to media supplemented with single nutrients or specific groups of nutrients. Of the 83 auxotrophs isolated, 65 required an inorganic source of nitrogen for growth. In addition, auxotrophs were isolated that required methionine (six), uracil (two), adenine (one), biotin (two), and nicotinic acid (two). (The number of isolates of each type is indicated in parentheses.) The nutrient requirements of five auxotrophs appeared complex and were not determined. A large proportion of the mutants requiring inorgainic fixed nitrogen was altered in the differentiation of heterocysts. The following morphological aberrancies were observed: abnormally high and abnormally low frequencies of heterocysts; thick, uneven heterocyst envelopes; incompletely developed pore regions; very distinct pore regions; and protoplasts separated from the envelope of the heterocyst. Spontaneously occurring, N2-fixing, prototrophic revertants of mutants with aberrant heterocysts have been isolated at a frequency of 2 X 10(-8) to 4 X 10(-8) of the cells plated. That most such revertants produced morphologically normal heterocysts is consisten with the idea that heterocysts play an essential role in aerobic N2 fixation.     

The Anabaena sp. strain PCC 7120 asr1734 gene encodes a negative regulator of heterocyst development

The novel asr1734 gene of Anabaena (Nostoc) sp. strain PCC 7120 inhibited heterocyst development when present in extra copies. Overexpression of asr1734 inhibited heterocyst development in several strains including the wild type and two strains that form multiple contiguous heterocysts (Mch phenotype): a PatS null mutant and a hetR(R223W) mutant. Overexpression of asr1734 also caused increased nblA messenger RNA levels, and increased loss of autofluorescence in vegetative cells throughout filaments after nitrogen or sulphur depletion. Unlike the wild type, an asr1734 knockout mutant formed 5% heterocysts after a nitrogen shift from ammonium to nitrate, and formed 15% heterocysts and a weak Mch phenotype after step-down to medium lacking combined nitrogen. After nitrogen step-down, the asr1734 mutant had elevated levels of ntcA messenger RNA. A green fluorescent protein reporter driven by the asr1734 promoter, P(asr1734)-gfp, was expressed specifically in differentiating proheterocysts and heterocysts after nitrogen step-down. Strains overexpressing asr1734 and containing P(hetR)-gfp or P(patS)-gfp reporters failed to show normal patterned upregulation 24 h after nitrogen step-down even though hetR expression was upregulated at 6 h. Apparent orthologues of asr1734 are found only in two other filamentous nitrogen-fixing cyanobacteria, Anabaena variabilis and Nostoc punctiforme.     

FraH is required for reorganization of intracellular membranes during heterocyst differentiation in Anabaena sp. strain PCC 7120

In the filamentous, heterocyst-forming cyanobacteria, two different cell types, the CO(2)-fixing vegetative cells and the N(2)-fixing heterocysts, exchange nutrients and regulators for diazotrophic growth. In the model organism Anabaena sp. strain PCC 7120, inactivation of fraH produces filament fragmentation under conditions of combined nitrogen deprivation, releasing numerous isolated heterocysts. Transmission electron microscopy of samples prepared by either high-pressure cryo-fixation or chemical fixation showed that the heterocysts of a ΔfraH mutant lack the intracellular membrane system structured close to the heterocyst poles, known as the honeycomb, that is characteristic of wild-type heterocysts. Using a green fluorescent protein translational fusion to the carboxyl terminus of FraH (FraH-C-GFP), confocal microscopy showed spots of fluorescence located at the periphery of the vegetative cells in filaments grown in the presence of nitrate. After incubation in the absence of combined nitrogen, localization of FraH-C-GFP changed substantially, and the GFP fluorescence was conspicuously located at the cell poles in the heterocysts. Fluorescence microscopy and deconvolution of images showed that GFP fluorescence originated mainly from the region next to the cyanophycin plug present at the heterocyst poles. Intercellular transfer of the fluorescent tracers calcein (622 Da) and 5-carboxyfluorescein (374 Da) was either not impaired or only partially impaired in the ΔfraH mutant, suggesting that FraH is not important for intercellular molecular exchange. Location of FraH close to the honeycomb membrane structure and lack of such structure in the ΔfraH mutant suggest a role of FraH in reorganization of intracellular membranes, which may involve generation of new membranes, during heterocyst differentiation.     

Identification of the active site of HetR protease and its requirement for heterocyst differentiation in the cyanobacterium Anabaena sp. strain PCC 7120

HetR is a serine-type protease required for heterocyst differentiation in heterocystous cyanobacteria under conditions of nitrogen deprivation. We have identified the active Ser residue of HetR from Anabaena sp. strain PCC 7120 by site-specific mutagenesis. By changing the S152 residue to an Ala residue, the mutant protein cannot be labeled by Dansyl fluoride, a specific serine-type protein inhibitor. The mutant protein showed no autodegradation in vitro. The mutant hetR gene was introduced into Anabaena strain 884a, a hetR mutant. The resultant strain, Anabaena strain S152A, could not form heterocysts under conditions of nitrogen deprivation even though the up-regulation of the mutant hetR gene was induced upon removal of combined nitrogen. The Anabaena strain 216, which carries a mutant hetR gene encoding S179N HetR and could not form heterocysts, also produced HetR protein upon induction. Sequence comparison shows that Ser152 is conserved in all cyanobacterial HetR. Immunoblotting was used to study HetR induction in both the wild-type and mutant strains. The amount of mutant HetR in strain S152A and in strain 216 increased continuously for 24 h after nitrogen step-down, while the amount of HetR in wild-type cells reached a maximum level within 6 h after nitrogen step-down. Our results show the Ser152 is the active site of HetR. The protease activity is required for heterocyst differentiation and might be needed for repression of HetR overproduction under conditions of nitrogen deprivation.     

Novel ATP-driven pathway of glycolipid export involving TolC protein

Upon depletion of combined nitrogen, N(2)-fixing heterocysts are formed from vegetative cells in the case of the filamentous cyanobacterium Anabaena sp. strain PCC 7120. A heterocyst-specific layer composed of glycolipids (heterocyst envelope glycolipids (HGLs)) that functions as an O(2) diffusion barrier is deposited over the heterocyst outer membrane and is surrounded by an outermost heterocyst polysaccharide envelope. Mutations in any gene of the devBCA operon or tolC result in the absence of the HGL layer, preventing growth on N(2) used as the sole nitrogen source. However, those mutants do not have impaired HGL synthesis. In this study, we show that DevBCA and TolC form an ATP-driven efflux pump required for the export of HGLs across the Gram-negative cell wall. By performing protein-protein interaction studies (in vivo formaldehyde cross-linking, surface plasmon resonance, and isothermal titration calorimetry), we determined the kinetics and stoichiometric relations for the transport process. For sufficient glycolipid export, the membrane fusion protein DevB had to be in a hexameric form to connect the inner membrane factor DevC and the outer membrane factor TolC. A mutation that impaired the ability of DevB to form a hexameric arrangement abolished the ability of DevC to recognize its substrate. The physiological relevance of a hexameric DevB is shown in complementation studies. We provide insights into a novel pathway of glycolipid export across the Gram-negative cell wall.     

THE HETEROCYSTS OF BLUE-GREEN ALGAE (MYXOPHYCEAE)

1. Heterocysts are found in many species of filamentous blue-green algae. They are cells of slightly larger size and with a more thickened wall than the vegetative cells. 2. Structural details of the heterocyst are: the presence of three additional wall layers, the absence of granules, sparse thylakoid network throughout, except at the poles where a dense coiling of membranes occurs. Other characters include the two pores at opposite poles ‘plugged’ with refractive material called the polar granule. 3. Peculiarities in the pigment composition of the heterocyst include an abundance of carotenoids and absence of phycobilins, and a short-wave form of chlorophyll a. 4. Unique glycolipids and an acyl lipid, not found in the vegetative cells of the algae or in other plant cells, are associated with the heterocyst. The glycolipids constitute the laminated layer of the wall and probably regulate diffusion of substances through it, whereas the acyl lipids are supposed to function as carriers and intermediates in the biosynthesis of the wall. 5. The heterocysts develop from vegetative cells, and the visible changes during differentiation include cell enlargement, synthesis of additional wall layers, disappearance of granules and reorientation and synthesis of the thylakoids. 6. Heterocysts are formed sequentially with characteristic cellular spacing during the growth of cultures in medium free from combined nitrogen. 7. Various sources of combined nitrogen inhibit heterocyst formation when supplied in the culture medium. Ammonium salts are among the most powerful inhibitors. Heterocysts are formed simultaneously and within a short period after transference of ammonia-grown non-heterocystous filaments to ammonia-free medium. 8. Incompletely differentiated heterocysts or proheterocysts are found in cultures grown in the presence of combined nitrogen. If two or more proheterocysts are close together generally a single one develops to maturity after a competitive interaction in medium free from combined nitrogen. This indicates that heterocyst formation is completed in two phases: phase I, synthesis and conservation of macromolecules, which takes place during growth in ammonia-containing medium: and phase 11, morphological differentiation of the heterocyst which is unaccompanied by growth in cell number. In the ammonia-free medium phase 11 quickly succeeds phase 1 and the whole process appears as a continuum. 9. Heterocyst formation shows a definite requirement for light. Red light favours heterocyst formation, whereas green and blue light do not. The effects of light seem to be mainly due to photosynthesis, although some effects may be morphogenetic. 10. Studies with metabolic inhibitors have revealed the involvement of photosynthesis, respiration and protein synthesis in heterocyst formation. Photosynthesis provides carbon skeletons, whereas ATP is most probably supplied by oxidative metabolism. 11. Various functions have been assigned to the heterocyst from time to time. Their role in akinete formation is suggested by (i) the formation of akinetes adjacent to the heterocysts and (ii) prevention of sporulation by detachment of the heterocysts from the vegetative cells (potential akinetes). Despite substantial evidence for such a role, it is not applicable to all akinete-forming genera. 12. Heterocysts are now widely believed to be the site of nitrogen fixation in blue-green algae. The main facts in favour of such a role are: (i) fixation of nitrogen by all heterocystous algae, (ii) inhibition of heterocyst formation by combined nitrogen and (iii) direct observations on acetylene reduction by isolated heterocysts. 13. Some non-heterocystous and unicellular algae, and vegetative cells of heterocystous algae fix nitrogen under microaerophilic conditions suggesting that absence of oxygen favours nitrogenase activity. Heterocysts lack the oxygen-evolving photo-system 11, possess oxidative enzymes, and reduce externally supplied tetrazolium salts - all indicating that they are the most suitable sites for harbouring nitrogenase in aerobic conditions. 14. Heterocysts probably originated in the Precambrian in response to the earth's changing environment and seem to be the first example of morphological differentiation in the plant kingdom.     

Effect on heterocyst differentiation of nitrogen fixation in vegetative cells of the cyanobacterium Anabaena variabilis ATCC 29413

Heterocysts are terminally differentiated cells of some filamentous cyanobacteria that fix nitrogen for the entire filament under oxic growth conditions. Anabaena variabilis ATCC 29413 is unusual in that it has two Mo-dependent nitrogenases; one, called Nif1, functions in heterocysts, while the second, Nif2, functions under anoxic conditions in vegetative cells. Both nitrogenases depended on expression of the global regulatory protein NtcA. It has long been thought that a product of nitrogen fixation in heterocysts plays a role in maintenance of the spaced pattern of heterocyst differentiation. This model assumes that each cell in a filament senses its own environment in terms of nitrogen sufficiency and responds accordingly in terms of differentiation. Expression of the Nif2 nitrogenase under anoxic conditions in vegetative cells was sufficient to support long-term growth of a nif1 mutant; however, that expression did not prevent differentiation of heterocysts and expression of the nif1 nitrogenase in either the nif1 mutant or the wild-type strain. This suggested that the nitrogen sufficiency of individual cells in the filament did not affect the signal that induces heterocyst differentiation. Perhaps there is a global mechanism by which the filament senses nitrogen sufficiency or insufficiency based on the external availability of fixed nitrogen. The filament would then respond by producing heterocyst differentiation signals that affect the entire filament. This does not preclude cell-to-cell signaling in the maintenance of heterocyst pattern but suggests that overall control of the process is not controlled by nitrogen insufficiency of individual cells.     

Synthesis of nitrogenase in mutants of the cyanobacterium Anabaena sp. strain PCC 7120 affected in heterocyst development or metabolism.

Mutants of Anabaena sp. strain PCC 7120 that are incapable of sustained growth with air as the sole source of nitrogen were generated by using Tn5-derived transposons. Nitrogenase was expressed only in mutants that showed obvious morphological signs of heterocyst differentiation. Even under rigorously anaerobic conditions, nitrogenase was not synthesized in filaments that were unable to develop heterocysts. These results suggest that competence to synthesize nitrogenase requires a process that leads to an early stage of visible heterocyst development and are consistent with the idea that synthesis of nitrogenase is under developmental control (J. Elhai and C. P. Wolk, EMBO J. 9:3379-3388, 1990). We isolated mutants in which differentiation was arrested at an intermediate stage of heterocyst formation, suggesting that differentiation proceeds in stages; those mutants, as well as mutants with aberrant heterocyst envelopes and a mutant with defective respiration, expressed active nitrogenase under anaerobic conditions only. These results support the idea that the heterocyst envelope and heterocyst respiration are required for protection of nitrogenase from inactivation by oxygen. In the presence of air, such mutants contained less nitrogenase than under anaerobic conditions, and the Fe-protein was present in a posttranslationally modified inactive form. We conclude that internal partial oxygen pressure sufficient to inactivate nitrogenase is insufficient to repress synthesis of the enzyme completely. Among mutants with an apparently intact heterocyst envelope and normal respiration, three had virtually undetectable levels of dinitrogenase reductase under all conditions employed. However, three others expressed oxygen-sensitive nitrogenase activity, suggesting that respiration and barrier to diffusion of gases may not suffice for oxygen protection of nitrogenase in these mutants; two of these mutants reduced acetylene to ethylene and ethane.     

Effect of controlled expression of the hetR gene on heterocyst formation in the filamentous cyanobacterium Anabaena sp. PCC 7120

hetR is a central regulatory gene inducing and possibly maintaining irreversible heterocyst differentiation in filamentous cyanobacteria. A plasmid was constructed which enabled IPTG-mediated, controlled expression of hetR from a p _tac promoter in Anabaena . When introduced into a heterocyst-deficient hetR mutant, induction led to massive formation of heterocysts in a medium free of combined nitrogen. In nitrate-containing cultures, induction elicited formation of only a few heterocysts, but led to nitrogen chlorosis in vegetative cells as evidenced from degradation of phycobiliproteins. Removal of the inducer IPTG caused chlorosis and death of the organisms in nitrate-free medium, but no reversal of heterocyst formation. This indicates that constant synthesis of HetR is not the (sole) reason for irreversibility of heterocyst formation.