Heterocyst formation in Anabaena

Heterocystous cyanobacteria grow as multicellular organisms with a distinct one-dimensional developmental pattern of single nitrogen-fixing heterocysts separated by approximately ten vegetative cells. Several genes have been identified that are required for heterocyst development and pattern formation. A key regulator, HetR, has been recently shown to be aserine-type protease.     

Heterocyst differentiation and tryptophan metabolism in the cyanobacterium Anabaena sp. CA

Anabaena sp. CA does not synthesize heterocysts or express nitrogenase activity when grown with nitrate as the nitrogen source. Heterocysts and nitrogenase are induced in such cultures by various tryptophan analogs. The effect does not require inhibition of de novo protein synthesis in the culture. It is restricted to tryptophan analogs only, and, more specifically, to those which can be incorporated into proteins. dl-7-Azatryptophan was effective at triggering both heterocysts and nitrogenase when incubated in the culture for only 1–2 h, even though 6–7 h was required for heterocysts to fully mature and nitrogenase activity to be expressed. Chloramphenicol completely negated this effect, supporting the idea that the analogs are either incorporated into protein themselves or trigger the synthesis of proteins which initiate complete development of mature heterocysts. Using toluene-permeabilized cells, we have shown that anthranilate synthetase, the first key enzyme in tryptophan biosynthesis, has glutamine-dependent activity. This activity can be effectively feedback inhibited by the various tryptophan analogs at concentrations which are also effective in triggering heterocyst differentiation. These data provide firm evidence for a link between tryptophan biosynthesis, nitrogenase synthesis, heterocyst differentiation, and primary ammonia assimilation.     

The rice field cyanobacteria Anabaena azotica and Anabaena sp. CH1 express vanadium-dependent nitrogenase

Anabaena azotica FACHB-118 and Anabaena sp. CH1, heterocystous cyanobacteria isolated from Chinese and Taiwanese rice fields, expressed vanadium-containing nitrogenase when under molybdenum deficiency. This is the second direct observation of an alternative nitrogenase in cyanobacteria. The vanadium nitrogenase-specific genes vnfDG are fused and clustered in a phylogenetic tree next to the corresponding genes of Methanosarcina. The expression of vnfH in cells cultured in Mo-free medium and of nifH in Mo-grown cells was shown for the first time by sequencing cDNA derived from cultures of A. azotica and Anabaena sp. CH1. The vnfH sequences clustered with that of Anabaena variabilis. The vnf genes were strongly transcribed only in cultures grown either in Mo-free medium, or in W-containing medium, but also weakly in Mo-containing medium. NifH was transcribed in all media. On-line measurements of acetylene reduction by Mo-free A. azotica cultures demonstrated that the V-nitrogenase was active. Ethane was formed continuously at a rate of 2.1% of that of ethylene. Acetylene reduction of cultures grown either with or without Mo had a high temperature optimum of 42.5°C. The uptake hydrogenase gene hupL was expressed in Mo-free medium concomitantly with vnfDG in A. azotica, Anabaena sp. CH1, and A. variabilis.     

D-erythrose supports nitrogenase activity in isolated Anabaena sp. strain 7120 heterocysts.

Among organic compounds tested for their ability to support nitrogenase activity in isolated heterocysts of Anabaena sp. strain 7120 under argon, D-erythrose (5 mM) was unique in supporting acetylene reduction at 10 times the control rates. Higher concentrations of D-erythrose exhibited substrate inhibition. At 50 kPa of H2, all concentrations of D-erythrose inhibited H2-supported acetylene reduction. The effects of D-erythrose on nitrogenase activity were explored. Erythrose enhanced 15N2 incorporation by heterocysts, but NADP+ did not enhance erythrose-supported acetylene reduction. H2 protected nitrogenase from O2 inactivation, but erythrose did not; erythrose did not counter protection by H2. Tests with inhibitors of electron transport showed that erythrose-supported acetylene reduction requires electron flow through ferredoxin, a b-type cytochrome, and a 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone-sensitive transfer agent whose electron flow is not mediated through the plastoquinone and Rieske iron protein.     

A mutant of Anabaena sp. CA with oxygen-sensitive nitrogenase activity.

Studies on the O₂ protection mechanism for nitrogenase in a mutant (PM10) of $\text{Anabaena}$ sp. CA indicated that the ability to protect nitrogenase from O₂ was functionally impaired. Growth rates of PM10 were substantially improved when cells were cultured under microaerobic conditions. Nitrogenase activity was totally inhibited by exposure to O₂ for 30 min; partial restoration of activity was attained when cell suspensions were subsequently made microaerobic. Experiments in which induction of nitrogenase activity was followed indicated that the synthesis of the O₂ protection mechanism was temporally separated from synthesis of heterocysts and nitrogenase.     

ppGpp Metabolism Is Involved in Heterocyst Development in the Cyanobacterium Anabaena sp. Strain PCC 7120

When deprived of a combined-nitrogen source in the growth medium, the filamentous cyanobacterium Anabaena sp. PCC 7120 (Anabaena) can form heterocysts capable of nitrogen fixation. The process of heterocyst differentiation takes about 20 to 24 h, during which extensive metabolic and morphological changes take place. Guanosine tetraphosphate (ppGpp) is the signal of the stringent response that ensures cell survival by adjusting major cellular activities in response to nutrient starvation in bacteria, and ppGpp accumulates at the early stage of heterocyst differentiation (J. Akinyanju, R. J. Smith, FEBS Lett. 107:173–176, 1979; J Akinyanju, R. J. Smith, New Phytol. 105:117–122, 1987). Here we show that all1549 (here designated rel_ana) in Anabaena, homologous to relA/spoT, is upregulated in response to nitrogen deprivation and predominantly localized in vegetative cells. The disruption of rel_ana strongly affects the synthesis of ppGpp, and the resulting mutant, all1549Ωsp/sm, fails to form heterocysts and to grow in the absence of a combined-nitrogen source. This phenotype can be complemented by a wild-type copy of rel_ana. Although the upregulation of hetR is affected in the mutant, ectopic overexpression of hetR cannot rescue the phenotype. However, we found that the mutant rapidly loses its viability, within a time window of 3 to 6 h, following the deprivation of combined nitrogen. We propose that ppGpp plays a major role in rebalancing the metabolic activities of the cells in the absence of the nitrogen source supply and that this regulation is necessary for filament survival and consequently for the success of heterocyst differentiation.     

Regulation of nitrogenase levels in Anabaena sp. ATCC 33047 and other filamentous cyanobacteria

Incubation in the dark of photoautotrophically grown N₂-fixing heterocystous cyanobacteria leads to a loss of nitrogenase activity. Original levels of nitrogenase activity are rapidly regained upon re-illumination of the filaments, in a process dependent on de novo protein synthesis. Ammonia, acting indirectly through some of its metabolic derivatives, inhibits the light-promoted development of nitrogenase activity in filaments of Anabaena sp. ATCC 33047 and several other cyanobacteria containing mature heterocysts. The ammonia-mediated control system is also operative in N₂-fixing filaments in the absence of any added source of combined nitrogen, with the ammonia resulting from N₂-fixation already partially inhibiting full expression of nitrogenase. High nitrogenase levels, about two-fold higher than those in normal N₂-fixing Anabaena sp. ATCC 33047, are found in cell suspensions which have been treated with the glutamine synthetase inhibitor l-methionine-d,l-sulfoximine or subjected to nitrogen starvation. Filaments treated in either way are insensitive to the ammonia-promoted inhibition of nitrogenase development, although this insensitivity is only transitory for the nitrogen-starved filaments, which become ammonia-sensitive once they regain their normal nitrogen status.Abbreviations Chl chlorophyll - EDTA ethylenediaminetetraacetic acid - MSX l-methionine-d,l-sulfoximine     

Monitoring Heterocyst Isolation in Anabaena PCC 7119

The purification of isolated and intact heterocysts is an essential step in the study and characterisation of their specific proteins. Therefore, a method for very successful heterocyst isolation from filamentous cyanobacteria, as monitored by measuring the presence of ferredoxin-NADP⁺ reductase activity during the isolation procedure has been developed.This is an improvement over the current lysozyme method in which damage could be caused to the heterocysts septum releasing soluble proteins. Frozen filaments should not be used for heterocysts isolation.     

Heterocyst development and diazotrophic metabolism in terminal respiratory oxidase mutants of the cyanobacterium Anabaena sp. strain PCC 7120

Heterocyst development was analyzed in mutants of the heterocyst-forming cyanobacterium Anabaena sp. strain PCC 7120 bearing inactivated cox2 and/or cox3 genes, encoding heterocyst-specific terminal respiratory oxidases. At the morphological level, the cox2 cox3 double mutant (strain CSAV141) was impaired in membrane reorganization involving the so-called honeycomb system that in the wild-type strain is largely or exclusively devoted to respiration, accumulated glycogen granules at conspicuously higher levels than the wild type (in both vegetative cells and heterocysts), and showed a delay in carboxysome degradation upon combined nitrogen deprivation. Consistently, chemical analysis confirmed higher accumulation of glycogen in strain CSAV141 than in the wild type. No impairment was observed in the formation of the glycolipid or polysaccharide layers of the heterocyst envelope, consistent with the chemical detection of heterocyst-specific glycolipids, or in the expression of the heterocyst-specific genes nifHDK and fdxH. However, nitrogenase activity under oxic conditions was impaired in strain CSAV135 (cox3) and undetectable in strain CSAV141 (cox2 cox3). These results show that these dedicated oxidases are required for normal development and performance of the heterocysts and indicate a central role of Cox2 and, especially, of Cox3 in the respiratory activity of the heterocysts, decisively contributing to protection of the N(2) fixation machinery against oxygen. However, in contrast to the case for other diazotrophic bacteria, expression of nif genes in Anabaena seems not to be affected by oxygen.     

Effects of D-erythrose on nitrogenase activity in whole filaments of Anabaena sp. strain 7120.

D-Erythrose, which has been shown to enhance nitrogenase activity (acetylene reduction) by isolated heterocysts, was studied for its effects on nitrogenase activity and nitrite uptake by whole filaments of Anabaena sp. strain 7120. D-Erythrose had little effect on acetylene reduction in the light; however, at a concentration of 10 mM, it could restore 3'-(3,4-dichlorophenyl)-1',1'-dimethyl urea-inhibited or dark-limited levels to light-supported levels. Sucrose, glucose, or fructose did not exhibit similar effects. D-Erythrose had little effect on nitrite uptake, an indirect measure of nitrite reductase activity by nitrate-grown whole filaments. It was concluded that erythrose effects were mediated by heterocysts and were therefore specific for nitrogenase.     

Heterocyst formation in cyanobacteria

When deprived of combined nitrogen, many filamentous cyanobacteria develop a one-dimensional pattern of specialised nitrogen-fixing cells, known as heterocysts. Recent years have seen the identification and characterisation of some of the key genes and proteins involved in heterocyst development and spacing, including the positive regulator HetR and the diffusible inhibitor PatS.     

Kinetics of synthesis of nitrogenase in batch and continuous culture of Anabaena flos-aquae

Summary Nitrogenase of Anabaena flos-aquae was inactivated by oxygen and recovery of activity was measured in batch and iron, phosphate and urea-limited continuous cultures. In batch culture, canavanine, chloramphenicol, methylamine, proflavine, puromycin and urea inhibited the recovery process. The rate of recovery of nitrogenase activity in continuous cultures was dependent on light intensity, concentration of urea, ammonium salts and nitrate, and independent of growth rate. Oxygen and urea caused an inactivation of nitrogenase in continuous cultures.     

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.     

Synthesis of nitrogenase and heterocysts by Anabaena sp. CA in the presence of high levels of ammonia.

Anabaena sp. CA fails to synthesize heterocysts and nitrogenase when grown with KNO3 as the nitrogen source. By contrast, both heterocysts and proheterocysts are synthesized in NH4Cl-containing media to a level nearly commensurate with cells grown in the absence of combined nitrogen. The growth rate of the organism in NH4Cl-containing media was similar to that obtained with KNO3 as the nitrogen source and was independent of the presence of N2 in the atmosphere. Thus, our results indicate that the organism assimilated nitrate and ammonium nitrogen equally well to meet the nitrogen requirements for growth. Moreover, in contrast to previous studies with other cyanobacteria, the repressor singal for heterocyst differentiation in Anabaena sp. CA is not derived from the metabolism of ammonia but appears to be involved with nitrate metabolism. Nitrogenase activity was partially expressed in NH4Cl-grown cultures. Increasing the level of nitrogenase activity to a value representative of a N2-grown culture required both the inhibition of ammonia assimilation and de novo protein synthesis. An increase in the number of mature heterocysts was not required. The fact that high levels of exogenous ammonia only partially repress the synthesis of proteins required for the maximum expression of nitrogenase activity in Anabaena sp. CA has important implications.     

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.