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.     

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.     

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.     

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.     

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.     

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.     

Improved eco-friendly recombinant Anabaena sp. strain PCC7120 with enhanced nitrogen biofertilizer potential

Photosynthetic, nitrogen-fixing Anabaena strains are native to tropical paddy fields and contribute to the carbon and nitrogen economy of such soils. Genetic engineering was employed to improve the nitrogen biofertilizer potential of Anabaena sp. strain PCC7120. Constitutive enhanced expression of an additional integrated copy of the hetR gene from a light-inducible promoter elevated HetR protein expression and enhanced functional heterocyst frequency in the recombinant strain. The recombinant strain displayed consistently higher nitrogenase activity than the wild-type strain and appeared to be in homeostasis with compatible modulation of photosynthesis and respiration. The enhanced combined nitrogen availability from the recombinant strain positively catered to the nitrogen demand of rice seedlings in short-term hydroponic experiments and supported better growth. The engineered strain is stable, eco-friendly, and useful for environmental application as nitrogen biofertilizer in paddy fields.     

patS minigenes inhibit heterocyst development of Anabaena sp. strain PCC 7120

The patS gene encodes a small peptide that is required for normal heterocyst pattern formation in the cyanobacterium Anabaena sp. strain PCC 7120. PatS is proposed to control the heterocyst pattern by lateral inhibition. patS minigenes were constructed and expressed by different developmentally regulated promoters to gain further insight into PatS signaling. patS minigenes patS4 to patS8 encode PatS C-terminal 4 (GSGR) to 8 (CDERGSGR) oligopeptides. When expressed by P(petE), P(patS), or P(rbcL) promoters, patS5 to patS8 inhibited heterocyst formation but patS4 did not. In contrast to the full-length patS gene, P(hepA)-patS5 failed to restore a wild-type pattern in a patS null mutant, indicating that PatS-5 cannot function in cell-to-cell signaling if it is expressed in proheterocysts. To establish the location of the PatS receptor, PatS-5 was confined within the cytoplasm as a gfp-patS5 fusion. The green fluorescent protein GFP-PatS-5 fusion protein inhibited heterocyst formation. Similarly, full-length PatS with a C-terminal hexahistidine tag inhibited heterocyst formation. These data indicate that the PatS receptor is located in the cytoplasm, which is consistent with recently published data indicating that HetR is a PatS target. We speculated that overexpression of other Anabaena strain PCC 7120 RGSGR-encoding genes might show heterocyst inhibition activity. In addition to patS and hetN, open reading frame (ORF) all3290 and an unannotated ORF, orf77, encode an RGSGR motif. Overexpression of all3290 and orf77 under the control of the petE promoter inhibited heterocyst formation, indicating that the RGSGR motif can inhibit heterocyst development in a variety of contexts.