Maintenance of heterocyst patterning in a filamentous cyanobacterium

In the absence of sufficient combined nitrogen, some filamentous cyanobacteria differentiate nitrogen-fixing heterocysts at approximately every 10th cell position. As cells between heterocysts grow and divide, this initial pattern is maintained by the differentiation of a single cell approximately midway between existing heterocysts. This paper introduces a mathematical model for the maintenance of the periodic pattern of heterocysts differentiated by Anabaena sp. strain PCC 7120 based on the current experimental knowledge of the system. The model equations describe a non-diffusing activator (HetR) and two inhibitors (PatS and HetN) that undergo diffusion in a growing one-dimensional domain. The inhibitors in this model have distinct diffusion rates and temporal expression patterns. These unique aspects of the model reflect recent experimental findings regarding the molecular interactions that regulate patterning in Anabaena. Output from the model is in good agreement with both the temporal and spatial characteristics of the pattern maintenance process observed experimentally.     

Characterization of HetR protein turnover in Anabaena sp. PCC 7120

The hetR gene plays an important role in heterocyst development and pattern formation in heterocystous cyanobacteria. The hetR gene from Anabaena sp. PCC 7120 was overexpressed in Escherichia coli. Antibodies raised against the recombinant HetR protein (rHetR) were used to characterize metabolism of the HetR of Anabaena sp. PCC 7120 in vivo. HetR was present at a low level when Anabaena sp. PCC 7120 was grown in the presence of combined nitrogen. Shifting from nitrogen repletion conditions to nitrogen depletion conditions led to a two fold increase of HetR in total cell extracts, and most of HetR was located in heterocysts. The amount of HetR in total cellular extracts increased rapidly after shifting to nitrogen depletion conditions and reached a maximum level 3 h after the shift. Isoelectrofocusing electrophoresis revealed that the native HetR had a more acidic isoelectric point than did rHetR. After combined nitrogen was added to the nitrogen-depleted cultures, the degradation of HetR depended on culture conditions: before heterocysts were fully developed, HetR was rapidly degraded; after heterocysts were fully developed, HetR was degraded much more slowly. The distribution of HetR in other species of cyanobacteria was also studied. Received: 24 June 1997 / Accepted: 5 December 1997     

Amplified expression of a transcriptional pattern formed during development of Anabaena

The cyanobacterium Anabaena responds to nitrogen deprivation by producing heterocysts, cells specialized for nitrogen fixation, at well-spaced intervals along its filaments. The gene hepA, required for heterocyst maturation, is expressed in response to nitrogen deprivation, prior to visible differentiation. A spatial pattern of hepA expression indistinguishable from the eventual pattern of heterocysts was made visible by fusing the hepA promoter to luxAB, which encodes bacterial luciferase. Because the resulting signal did not greatly exceed instrumental background, T7 RNA polymerase was used to increase luminescence. The hepA promoter was fused to the gene for that polymerase, and a promoter recognized by that polymerase was fused to luxAB. Filaments containing these two fusions showed spaced luminescing cells many hours before differentiation became discernible morphologically.     

The hetZ gene indirectly regulates heterocyst development at the level of pattern formation in Anabaena sp. strain PCC 7120

Multicellular development requires the careful orchestration of gene expression to correctly create and position specialized cells. In the filamentous cyanobacterium Anabaena sp. strain PCC 7120, nitrogen‐fixing heterocysts are differentiated from vegetative cells in a reproducibly periodic and physiologically relevant pattern. While many genetic factors required for heterocyst development have been identified, the role of HetZ has remained unclear. Here, we present evidence to clarify the requirement of hetZ for heterocyst production and support a model where HetZ functions in the patterning stage of differentiation. We show that a clean, nonpolar deletion of hetZ fails to express the developmental genes hetR, patS, hetP and hetZ correctly and fails to produce heterocysts. Complementation and overexpression of hetZ in a hetP mutant revealed that hetZ was incapable of bypassing hetP, suggesting that it acts upstream of hetP. Complementation and overexpression of hetZ in a hetR mutant, however, demonstrated bypass of hetR, suggesting that it acts downstream of hetR and is capable of bypassing the need for hetR for differentiation irrespective of nitrogen status. Finally, protein–protein interactions were observed between HetZ and HetR, Alr2902 and HetZ itself. Collectively, this work suggests a regulatory role for HetZ in the patterning phase of cellular differentiation in Anabaena.     

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.     

Consequences of the iron-dependent formation of ferredoxin and flavodoxin on photosynthesis and nitrogen fixation on Anabaena strains

Iron-dependent formation of ferredoxin and flavodoxin was determined in Anabaena ATCC 29413 and ATCC 29211 by a FPLC procedure. In the first species ferredoxin is replaced by flavodoxin at low iron levels in the vegetative cells only. In the heterocysts from Anabaena ATCC 29151, however, flavodoxin is constitutively formed regardless of the iron supply.Replacement of ferredoxin by flavodoxin had no effect on photosynthetic electron transport, whereas nitrogen fixation was decreased under low iron conditions. As ferredoxin and flavodoxin exhibited the same K_m values as electron donors to nitrogenase, an iron-limited synthesis of active nitrogenase was assumed as the reason for inhibited nitrogen fixation. Anabaena ATCC 29211 generally lacks the potential to synthesize flavodoxin. Under iron-starvation conditions, ferredoxin synthesis is limited, with a negative effect on photosynthetic oxygen evolution.     

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.     

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     

Calcium-dependent protease of the cyanobacterium Anabaena: molecular cloning and expression of the gene in Escherichia coli, sequencing and site-directed mutagenesis

Summary It has been suggested that a calcium-dependent intracellular protease of the cyanobacterium, Anabaena sp., participates in the differentiation of heterocysts, cells that are specialized for fixation of N₂. Clones of the structural gene (designated prcA) for this protease from Anabaena variabilis strain ATCC 29413 and Anabaena sp. strain PCC 7120 were identified via their expression in Escherichia coli. The prcA gene from A. variabilis was sequenced. The genes of both strains, mutated by insertion of a drug resistance cassette, were returned to these same strains of Anabaena on suicide plasmids. The method of sacB-mediated positive selection for double recombinants was used to achieve replacement of the wild-type prcA genes by the mutated forms. The resulting mutants, which lacked Ca²⁺-dependent protease activity, were not impaired in heterocyst formation and grew on N₂ as sole nitrogen source.     

MSX-resistant mutants of Anabaena 7120 with derepressed heterocyst development and nitrogen fixation

To investigate the role of ammonium-assimilating enzyme in heterocyst differentiation, pattern formation and nitrogen fixation, MSX-resistant and GS-impaired mutants of Anabaena 7120 were isolated using transposon (Tn5-1063) mutagenesis. Mutant Gs1 and Gs2 (impaired in GS activity) exhibited a similar rate of nitrogenase activity compared to that of the wild type under dinitrogen aerobic conditions in the presence and absence of MSX. Filaments of Gs1 and Gs2 produced heterocysts with an evenly spaced pattern in N₂-grown conditions, while addition of MSX altered the interheterocyst spacing pattern in wild type as well as in mutant strains. The wild type showed complete repression of heterocyst development and nitrogen fixation in the presence of NO₃ ^– or NH₄ ⁺, whereas the mutants Gs1 and Gs2 formed heterocysts and fixed nitrogen in the presence of NO₃ ^– and NH₄ ⁺. Addition of MSX caused complete inhibition of glutamine synthetase activity in wild type but Gs1 and Gs2 remained unaffected. These results suggest that glutamine but not ammonium is directly involved in regulation of heterocyst differentiation, interheterocyst spacing pattern and nitrogen fixation in Anabaena.     

Expression of Anabaena ferredoxin genes in Escherichia coli

The genes for ferredoxin from heterocysts (fdx H) and vegetative cells (pet F) of Anabaena sp. strain 7120 were subcloned into plasmid pUC 18/19. Both genes were expressed in Escherichia coli at high levels (10% of total protein). Pet F could be expressed from its own promoter. The ferredoxins were correctly assembled to the holoprotein. Heterocyst ferredoxin was purified from E. coli extracts on a large scale. Its biochemical and biophysical properties were identical to those of the authentic ferredoxin, isolated from Anabaena heterocysts.This paper is dedicated to Prof. A. Trebst on the occasion of his 60th birthday.     

Overexpression of SepJ alters septal morphology and heterocyst pattern regulated by diffusible signals in Anabaena

Filamentous, N₂‐fixing, heterocyst‐forming cyanobacteria grow as chains of cells that are connected by septal junctions. In the model organism Anabaena sp. strain PCC 7120, the septal protein SepJ is required for filament integrity, normal intercellular molecular exchange, heterocyst differentiation, and diazotrophic growth. An Anabaena strain overexpressing SepJ made wider septa between vegetative cells than the wild type, which correlated with a more spread location of SepJ in the septa as observed with a SepJ–GFP fusion, and contained an increased number of nanopores, the septal peptidoglycan perforations that likely accommodate septal junctions. The septa between heterocysts and vegetative cells, which are narrow in wild‐type Anabaena, were notably enlarged in the SepJ‐overexpressing mutant. Intercellular molecular exchange tested with fluorescent tracers was increased for the SepJ‐overexpressing strain specifically in the case of calcein transfer between vegetative cells and heterocysts. These results support an association between calcein transfer, SepJ‐related septal junctions, and septal peptidoglycan nanopores. Under nitrogen deprivation, the SepJ‐overexpressing strain produced an increased number of contiguous heterocysts but a decreased percentage of total heterocysts. These effects were lost or altered in patS and hetN mutant backgrounds, supporting a role of SepJ in the intercellular transfer of regulatory signals for heterocyst differentiation.     

Rearrangements of nitrogen fixation (nif) genes in the heterocystous cyanobacteria

In the vegetative cells of heterocystous cyanobacteria, such asAnabaena, two Operons harbouring the nitrogen fixaton (nif) genes contain two separate intervening DNA elements resulting in the dispersion of genes and impaired gene expression. A 11 kb element disrupts thenifD gene in thenifH, D-K operon. It contains a 11 bp sequence (GGATTACTCCG) directly repeated at its ends and harbours a gene,xisA, which encodes a site-specific recombinase. A large 55 kb element interrupts thefdxN gene in thenifB fdxN-nifS-nifU operon. It contains two 5 bp direct repeats (TATTC) at its ends and accommodates at least one gene,xisF, which encodes another site-specific recombinase. During heterocyst differentiation both the discontinuities are precisely excised by two distinct site-specific recombination events. One of them is brought about by the XisA protein between the 11 bp direct repeats. The second one is caused by the XisF protein and occurs between the 5 bp direct repeats. As a consequence the 11kb and 55 kb elements are removed from the chromosome as circles and functionalnif Operons are created. Nitrogenase proteins are then expressed from the rearranged genes in heterocysts and aerobic nitrogen fixation ensues. How these elements intruded thenif genes and how and why are they maintained in heterocystous cyanobacteria are exciting puzzles engaging considerable research effort currently. The unique developmental regulation of these gene rearrangements in heterocystous cyanobacteria is discussed.     

Localization of a multifunctional chaperonin (GroEL protein) in nitrogen-fixing Anabaena PCC 7120

The occurrence and distribution of a multifunctional chaperonin-60 (cpn60), the GroEL protein, was demonstrated in the cyanobacterium Anabaena PCC 7120 by using a rabbit anti-GroEL (Escherichia coli) antibody. Western-blot analysis showed a distinct cross-reaction with a protein of approx. 65 kilodaltons, analogous to the Mr of the E. coli homologue. Immunocyto-chemical studies of vegetative cells showed that a chaperonin was localized in both vegetative cells and heterocysts. In vegetative cells cpn60 was primarily detected both in the carboxysomes, and in the cytoplasm, though mainly in the thylakoid region of the latter. In heterocysts, specialized cells for nitrogen fixation, the cpn60 label was prominent and was evenly distributed throughout the cell. These results support recent findings that chaperonins are multifunctional proteins, and extend those findings by demonstrating the occurrence of cpn60 in a prokaryotic cyanobacterium and by raising the possibility of the involvement of this chaperonin in the assembly of heterocystous proteins.Abbreviations cpn60 chaperonin-60 - Mr relative molecular mass - Rubisco ribulose-1,5-bisphosphate carboxylase/oxygenase     

Proteome Analysis of Enriched Heterocysts from Two Hydrogenase Mutants from Anabaena sp. PCC 7120

Cyanobacteria are oxygenic photosynthetic prokaryotes and play a crucial role in the Earth's carbon and nitrogen cycles. The photoautotrophic cyanobacterium Anabaena sp. PCC 7120 has the ability to fix atmospheric nitrogen in heterocysts and produce hydrogen as a byproduct through a nitrogenase. In order to improve hydrogen production, mutants from Anabaena sp. PCC 7120 are constructed by inactivation of the uptake hydrogenase (ΔhupL) and the bidirectional hydrogenase (ΔhoxH) in previous studies. Here the proteomic differences of enriched heterocysts between these mutants cultured in N₂‐fixing conditions are investigated. Using a label‐free quantitative proteomics approach, a total of 2728 proteins are identified and it is found that 79 proteins are differentially expressed in the ΔhupL and 117 proteins in the ΔhoxH variant. The results provide for the first time comprehensive information on proteome regulation of the uptake hydrogenase and the bidirectional hydrogenase, as well as systematic data on the hydrogen related metabolism in Anabaena sp. PCC 7120.