Zn(2+) site engineering at the oligomeric interface of the dopamine transporter

The hetN gene plays an important role in heterocyst differentiation and pattern formation. An immunoblotting study showed that the hetN gene in Anabaena sp. PCC 7120 was expressed in vegetative cells grown with combined nitrogen. After a switch to a medium without combined nitrogen, hetN expression first declined and was then followed by a rapid increase in its product, HetN, which was only present in mature heterocysts. HetN is located on both thylakoid membranes and plasma membranes as determined by immunoblotting using purified membranes. Overexpression of hetN completely prevented hetR up-regulation under nitrogen-deprivation conditions, suggesting that its role in pattern control may depend on its inhibition of hetR expression.     

The RGSGR amino acid motif of the intercellular signalling protein, HetN, is required for patterning of heterocysts in Anabaena sp. strain PCC 7120

Nitrogen-fixing heterocysts are arranged in a periodic pattern on filaments of the cyanobacterium Anabaena sp. strain PCC 7120 under conditions of limiting combined nitrogen. Patterning requires two inhibitors of heterocyst differentiation, PatS and HetN, which work at different stages of differentiation by laterally suppressing levels of an activator of differentiation, HetR, in cells adjacent to source cells. Here we show that the RGSGR sequence in the 287-amino-acid HetN protein, which is shared by PatS, is critical for patterning. Conservative substitutions in any of the five amino acids lowered the extent to which HetN inhibited differentiation when overproduced and altered the pattern of heterocysts in filaments with an otherwise wild-type genetic background. Conversely, substitution of amino acids comprising the putative catalytic triad of this predicted reductase had no effect on inhibition or patterning. Deletion of putative domains of HetN suggested that the RGSGR motif is the primary component of HetN required for both its inhibitory and patterning activity, and that localization to the cell envelope is not required for patterning of heterocysts. The intercellular signalling proteins PatS and HetN use the same amino acid motif to regulate different stages of heterocyst patterning.     

Inactivation of patS and hetN causes lethal levels of heterocyst differentiation in the filamentous cyanobacterium Anabaena sp. PCC 7120

Summary In the filamentous cyanobacterium Anabaena sp. PCC 7120 patS and hetN suppress the differentiation of vegetative cells into nitrogen-fixing heterocysts to establish and maintain a pattern of single heterocysts separated by approximately 10 undifferentiated vegetative cells. Here we show that the patS- and hetN-dependent suppression pathways are the only major factors that prevent vegetative cells from differentiating into heterocysts when a source of ammonia is not present. The patS and hetN pathways are independent of each other, and inactivation of both patS and hetN leads to differentiation of almost all cells of a filament in the absence of a source of fixed nitrogen, compared with approximately 9% in the wild type. Complete differentiation of filaments also occurs when nitrate is supplied as a source of fixed nitrogen, conditions that do not induce differentiation of wild-type filaments. However, ammonia is still capable of suppressing differentiation. The percentage of cells that differentiate into heterocysts appears to be a function of time when a source of fixed nitrogen is absent or a function of growth phase when nitrate is supplied. Although differentiation proceeds unchecked in the absence of patS and hetN expression, differentiation is asynchronous and non-random.     

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.     

Role for hetC in the transition to a nondividing state during heterocyst differentiation in Anabaena sp

Nitrogen-deprived filaments of wild-type or hetC Anabaena sp. produce respectively, at semiregular intervals, heterocysts and weakly fluorescent cells. Unlike heterocysts, the latter cells can divide and elongate, producing a pattern of spaced series of small cells. Because a hetR::gfp fusion is expressed most strongly in the small cells, we propose that these small cells represent a very early stage of heterocyst differentiation. hetC::gfp is expressed most strongly in proheterocysts and heterocysts.     

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.     

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.     

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.     

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.     

Analysis of a Het- mutation in Anabaena sp. strain PCC 7120 implicates a secondary metabolite in the regulation of heterocyst spacing.

Transposon-generated mutant N10 of Anabaena sp. strain PCC 7120 has a Het- phenotype (A. Ernst, T. Black, Y. Cai, J.-M. Panoff, D. N. Tiwari, and C. P. Wolk, J. Bacteriol. 174:6025-6032, 1992). Reconstruction of the transposon mutation reproduced a Het- phenotype, but reconstructions with other insertions at the position of the transposon produced strains that form multiple contiguous heterocysts. Sequence analysis around the site of insertion of the transposon showed that the insertion lies within the 5' end of an 861-bp open reading frame (ORF) (hetN). The product of translation of hetN (HetN) shows extensive similarity to NAD(P)H-dependent oxidoreductases that are involved in biosyntheses of fatty acids, poly-beta-hydroxybutyrate, nod factor, and polyketides. A second, 1,518-bp ORF (hetM) that ends 556 bp 5' from the start of hetN appears to encode a protein that has at least two functional domains: its amino terminus is similar to an acyl carrier protein, while its central portion is similar to domains of proteins that perform reductive reactions. A third, 711-bp ORF (hetI) encoded on the opposite strand ends 42 bp away from the 3' end of hetN. The protein encoded by hetI, HetI, is similar to Sfp from Bacillus subtilis and EntD from Escherichia coli, proteins that are required for the biosynthesis or export of cyclic peptides. Clones from a lambda-EMBL3 library that contain the wild-type DNA for hetN do not complement the hetN::Tn5-1063 mutation in N10. The presence of hetN, as the only ORF, on a replicating plasmid suppresses heterocyst formation in wild-type cells, whereas the additional presence of hetI alleviates this effect.     

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.     

Tracing the path of a prokaryotic paracrine signal

Filamentous heterocyst‐forming cyanobacteria are a beautiful example of prokaryotic multicellularity. The filaments can achieve simultaneous nitrogen fixation and oxygenic photosynthesis by cooperation between two cell types: the photosynthetic vegetative cells and the nitrogen‐fixing heterocysts. The multicellular features exhibited by the system include differentiation of different cell types, metabolic interdependence and even pattern formation, as the spacing of heterocysts along the filament is non‐random. Recent years have seen exciting progress both in understanding the control of heterocyst differentiation, and also in understanding the function of ‘septal junctions’: an array of pore‐like structures at the cell junctions that allow intercellular communication by facilitating the diffusion of small molecules from cell to cell. A new report by Rivers et al. (2014) makes the connection between pattern formation and intercellular communication by showing that a mutation that partially disables the septal junctions leads to a decrease in the range of a signal dependent on the HetN protein that is one of the factors controlling heterocyst spacing. This suggests that the signal travels from cell to cell by diffusion through the septal junctions, opening the door to quantitative understanding of the mechanism that controls heterocyst spacing in filamentous cyanobacteria.     

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].     

Heterocyst-specific expression of patB,a gene required for nitrogen fixation in Anabaena sp. strain PCC 7120

The patB gene product is required for growth and survival of the filamentous cyanobacterium Anabaena sp. strain PCC 7120 in the absence of combined nitrogen. A patB::gfp fusion demonstrated that this gene is expressed exclusively in heterocysts. patB mutants have a normal initial pattern of heterocyst spacing along the filament but differentiate excess heterocysts after several days in the absence of combined nitrogen. Expression of hetR and patS, two critical regulators of the heterocyst development cascade, are normal for patB mutants, indicating that patB acts downstream of them in the differentiation pathway. A patB deletion mutant suffers an almost complete cessation of growth and nitrogen fixation within 24 h of combined nitrogen removal. In contrast, a new PatB mutant that is defective in its N-terminal ferredoxin domain, or a previously described mutant that has a frameshift removing its C-terminal helix-turn-helix domain, grows very slowly and differentiates multiple contiguous heterocysts under nitrogen-deficient conditions.     

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.