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.     

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     

Two mutations that block heterocyst differentiation have different effects on akinete differentiation in Nostoc ellipsosporum

Evident differentiation of vegetative cells into hetero-cysts in Anabaena sp. strain PCC 7120 is prevented by Insertions in genes hetR and hetP. Nostoc ellipsosporum possesses single copies of genes that hybridize with hetR and hetP. In mutant NE2 of N. ellipsosporum, in which hetR is interrupted by an insert, and in a double recombinant of wild-type N. ellipsosporum with a plasmid that bears an interrupted copy of hetR, neither heterocysts nor akinetes are formed. When an intact copy of hetR from Anabaena sp. strain PCC 7120 was added to NE2 the ability to form both heterocysts and akinetes was restored, in contrast to the hetR mutant, a hetP mutant of N. ellipsosporum could form akinetes, but heterocyst formation was blocked. Use of luxAB, encoding luciferase, as a reporter, and use of luxC, luxD and luxE to generate aldehyde (a substrate for the luciferase reaction), permitted visualization of the expression of hetR at the level of single cells; hetR was expressed in akinetes.     

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].     

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.     

Mutagenesis of hetR reveals amino acids necessary for HetR function in the heterocystous cyanobacterium Anabaena sp. strain PCC 7120

HetR is the master regulator of heterocyst differentiation in the filamentous cyanobacterium Anabaena sp. strain PCC 7120. Genetic selection was used to identify 33 amino acid substitutions in HetR that reduced the proportion of cells undergoing heterocyst differentiation to less than 2%. Conservative substitutions in the wild-type HetR protein revealed three mutations that dramatically reduced the amount of heterocyst differentiation when the mutant allele was present in place of the wild-type allele on a replicating plasmid in a mutant lacking hetR on the chromosome. An H69Y substitution resulted in heterocyst formation among less than 0.1% of cells, and D17E and G36A substitutions resulted in a Het- phenotype, compared to heterocyst formation among approximately 25% of cells with the wild-type hetR under the same conditions. The D17E substitution prevented DNA binding activity exhibited by wild-type HetR in mobility shift assays, whereas G36A and H69Y substitutions had no affect on DNA binding. D17E, G36A, and H69Y substitutions also resulted in higher levels of the corresponding HetR protein than of the wild-type protein when each was expressed from an inducible promoter in a hetR deletion strain, suggesting an effect on HetR protein turnover. Surprisingly, C48A and S152A substitutions, which were previously reported to result in a Het- phenotype, were found to have no effect on heterocyst differentiation or patterning when the corresponding mutations were introduced into an otherwise wild-type genetic background in Anabaena sp. strain PCC 7120. The clustering of mutations that satisfied the positive selection near the amino terminus suggests an important role for this part of the protein in HetR function.     

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.     

The role of HetN in maintenance of the heterocyst pattern in Anabaena sp. PCC 7120

The gene hetN encodes a putative oxidoreductase that is known to suppress heterocyst differentiation when present on a multicopy plasmid in Anabaena sp. PCC 7120. To mimic the hetN null phenotype and to examine where HetN acts in the regulatory cascade that controls heterocyst differentiation, we replaced the native chromosomal hetN promoter with the copper-inducible petE promoter. In the presence of copper, heterocyst formation was suppressed in undifferentiated filaments. When hetN expression was turned off by transferring cells to media lacking copper, the filaments initially displayed the wild-type pattern of single heterocysts but, 48 h after the induction of heterocyst formation, a pattern of multiple contiguous heterocysts predominated. Suppression of heterocyst formation by HetN appears to occur both upstream and downstream of the positive regulator HetR: overexpression of hetN in undifferentiated filaments prevents the wild-type pattern of hetR expression as well as the multiheterocyst phenotype normally observed when hetR is expressed from an inducible promoter. Green fluorescent protein fusions show that the expression of hetN in wild-type filaments normally occurs primarily in heterocysts. We propose that HetN is normally involved in the maintenance of heterocyst spacing after the initial heterocyst pattern has been established, but ectopic expression of hetN can also block the initial establishment of the pattern.     

Overexpression of pknE blocks heterocyst development in Anabaena sp. strain PCC 7120

The upstream intergenic regions for each of four genes encoding Ser/Thr kinases, all2334, pknE (alr3732), all4668, and all4838, were fused to a gfpmut2 reporter gene to determine their expression during heterocyst development in the cyanobacterium Anabaena (Nostoc) sp. strain PCC 7120. P(pknE)-gfp was upregulated after nitrogen step-down and showed strong expression in differentiating cells. Developmental regulation of pknE required a 118-bp upstream region and was abolished in a hetR mutant. A pknE mutant strain had shorter filaments with slightly higher heterocyst frequency than did the wild type. Overexpression of pknE from its native promoter inhibited heterocyst development in the wild type and in four mutant backgrounds that overproduce heterocysts. Overexpression of pknE from the copper-inducible petE promoter did not completely inhibit heterocyst development but caused a 24-h delay in heterocyst differentiation and cell bleaching 4 to 5 days after nitrogen step-down. Strains overexpressing pknE and containing P(hetR)-gfp or P(patS)-gfp reporters failed to show developmental regulation of the reporters and had undetectable levels of HetR protein. Genetic epistasis experiments suggest that overexpression of pknE blocks HetR activity or downstream regulation.     

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.