CiteULike: cbg's biochempaper

Nitrogen control in cyanobacteria.

A Herrero — 2008-05-09T14:40:27-00:00

Journal of bacteriology, Vol. 183, No. 2. (January 2001), pp. 411-425.

Mutual dependence of the expression of the cell differentiation regulatory protein HetR and the global nitrogen regulator NtcA during heterocyst development

Alicia — 2008-05-09T14:33:23-00:00

Molecular Microbiology, Vol. 44, No. 5. (2002), pp. 1377-1385.

Summary Heterocyst differentiation in the cyanobacterium Anabaena sp. strain PCC 7120 depends on both the global nitrogen regulator NtcA and the cell differentiation regulatory protein HetR, and induction of hetR upon nitrogen step-down depends on NtcA. The use of two out of the four transcription start points (tsps) described for the hetR gene (those located at positions -728 and -271) was found to be dependent on NtcA, and the use of the tsp located at position -271 was also dependent on HetR. Thus, autoregulation of hetR could take place via the activation of transcription from this tsp. Expression of ntcA in nitrogen-fixing cultures was higher than in cells growing in the presence of ammonium or nitrate, and high expression of ntcA under nitrogen deficiency resulted from an increased use of tsps located at positions -180 and -49. The induction of the use of these tsps did not take place in ntcA or hetR mutant strains. These results indicate a mutual dependency in the induction of the regulatory genes hetR and ntcA that takes place in response to nitrogen step-down in Anabaena cells. Expression of the hetC gene, which is also involved in the early steps of heterocyst differentiation, from its NtcA-dependent tsp was, however, not dependent on HetR.

Complete Genomic Sequence of the Filamentous Nitrogen-fixing Cyanobacterium Anabaena sp. Strain PCC 7120

Takakazu Kaneko — 2008-05-09T12:37:25-00:00

DNA Res, Vol. 8, No. 5. (1 January 2001), pp. 205-213.

The nucleotide sequence of the entire genome of a filamentous cyanobacterium, Anabaena sp. strain PCC 7120, was determined. The genome of Anabaena consisted of a single chromosome (6,413,771 bp) and six plasmids, designated pCC7120alpha (408,101 bp), pCC7120beta (186,614 bp), pCC7120gamma (101,965 bp), pCC7120delta (55,414 bp), pCC7120epsilon (40,340 bp), and pCC7120zeta (5,584 bp). The chromosome bears 5368 potential protein-encoding genes, four sets of rRNA genes, 48 tRNA genes representing 42 tRNA species, and 4 genes for small structural RNAs. The predicted products of 45% of the potential protein-encoding genes showed sequence similarity to known and predicted proteins of known function, and 27% to translated products of hypothetical genes. The remaining 28% lacked significant similarity to genes for known and predicted proteins in the public DNA databases. More than 60 genes involved in various processes of heterocyst formation and nitrogen fixation were assigned to the chromosome based on their similarity to the reported genes. One hundred and ninety-five genes coding for components of two-component signal transduction systems, nearly 2.5 times as many as those in Synechocystis sp. PCC 6803, were identified on the chromosome. Only 37% of the Anabaena genes showed significant sequence similarity to those of Synechocystis, indicating a high degree of divergence of the gene information between the two cyanobacterial strains. 10.1093/dnares/8.5.205

HetR homodimer is a DNA-binding protein required for heterocyst differentiation, and the DNA-binding activity is inhibited by PatS

Xu Huang — 2008-05-09T11:37:24-00:00

Proceedings of the National Academy of Sciences, Vol. 101, No. 14. (6 April 2004), pp. 4848-4853.

HetR plays a key role in regulation of heterocyst differentiation. When the Cys-48 residue of the HetR from Anabaena sp. PCC 7120 was replaced with an Ala residue, the mutant HetR (HetRC48A) could not dimerize, indicating that HetR forms a homodimer through a disulfide bond. The Anabaena strain C48, containing the hetRc48a gene, could not produce HetR homodimer and failed to form heterocyst. We show that HetR is a DNA-binding protein and that its homodimerization is required for the DNA binding. HetR binds the promoter regions of hetR, hepA, and patS, suggesting a direct control of the expression of these genes by HetR. We present evidence that shows that the up-regulation of patS and hetR depends on DNA binding by HetR dimer. The pentapeptide RGSGR, which is present at the C terminus of PatS and blocks heterocyst formation, inhibits the DNA binding of HetR and prevents hetR up-regulation. 10.1073/pnas.0400429101

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

Sean Callahan — 2008-05-09T10:41:58-00:00

Molecular Microbiology, Vol. 40, No. 4. (2001), pp. 941-950.

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.

Expression of hetN during heterocyst differentiation and its inhibition of hetR up-regulation in the cyanobacterium Anabaena sp. PCC 7120

Bin Li — 2008-05-09T07:38:14-00:00

FEBS Letters, Vol. 517, No. 1-3. (24 April 2002), pp. 87-91.

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.

Heterocyst Pattern Formation Controlled by a Diffusible Peptide

Ho-Sung Yoon — 2008-05-09T00:36:51-00:00

Science, Vol. 282, No. 5390. (30 October 1998), pp. 935-938.

10.1126/science.282.5390.935

Characterization of HetR protein turnover in Anabaena sp. PCC 7120

Ruanbao Zhou — 2008-05-08T22:35:08-00:00

Archives of Microbiology, Vol. 169, No. 5. (18 April 1998), pp. 417-426.

Abstract 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.

Requirement of the regulatory protein NtcA for the expression of nitrogen assimilation and heterocyst development genes in the cyanobacterium Anabaena sp. PCC7120

Jose Frias — 2008-05-08T22:16:25-00:00

Molecular Microbiology, Vol. 14, No. 4. (1994), pp. 823-832.

Summary The cyanobacterial ntcA gene encodes a DNA-binding protein that belongs to the Crp family of bacterial transcriptional regulators. In this work, we describe the isolation of an ntcA insertional mutant of the dinitrogen-fixing, heterocyst-forming cyanobacterium Anabaena sp. PCC 7120. The Anabaena ntcA mutant was able to use ammonium as a source of nitrogen for growth, but was unable to assimilate atmospheric nitrogen (dinitrogen) or nitrate. Nitrogenase and enzymes of the nitrate reduction system were not synthesized in the ntcA mutant under derepressing conditions, and glutamine synthetase levels were lower in the mutant than in the wild-type strain. In the ntcA mutant, in response to removal of ammonium, accumulation of mRNA of the genes encoding nitrogenase (nifHDK), nitrite reductase (nir, the first gene of the nitrate assimilation operon), and glutamine synthetase (glnA) was not observed. A transcription start point of the Anabaena glnA gene (corresponding to RNA1), that has been shown to be used preferentially after nitrogen step-down, was not used in the ntcA insertional mutant. Heterocyst development (which is necessary for the aerobic fixation of dinitrogen) and induction of hetR (a regulatory gene that is required for heterocyst development) were also impaired in the ntcA mutant. These results showed that the ntcA gene product, NtcA, is required in Anabaena sp. PCC 7120 for the expression of genes encoding proteins involved in the assimilation of nitrogen sources alternative to ammonium including dinitrogen and nitrate, and that the process of heterocyst development is also controlled by NtcA.

Identification of the active site of HetR protease and its requirement for heterocyst differentiation in the cyanobacterium Anabaena sp. strain PCC 7120.

Y Dong — 2008-05-08T21:38:25-00:00

Journal of bacteriology, Vol. 182, No. 6. (March 2000), pp. 1575-1579.

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.

Spatial expression and autoregulation of hetR, a gene involved in the control of heterocyst development in Anabaena

Todd Black — 2008-05-08T19:34:10-00:00

Molecular Microbiology, Vol. 9, No. 1. (1993), pp. 77-84.

Summary The spatially patterned differentiation of hetero-cysts in the filamentous cyanobacterium Anabaena requires a functional hetR gene. Transcriptional fusions to luxAB show that hetR is transcribed at a low level throughout the filament when Anabaena is grown with combined nitrogen, and that induction of the gene begins within 2 h following nitrogen deprivation. By 3.5 h, induction is localized to spaced foci. By 6h, there is an overall induction of at least threefold in whole cultures, reflecting at least a 20-fold increase within spatially separated cells. The induction requires the presence of a functional hetR gene, indicating that hetR is autoregulatory. Full induction of a heterocyst structural gene, hepA, also requires a functional hetR locus.

Evidence that HetR protein is an unusual serine-type protease

Ruanbao Zhou — 2008-05-08T19:08:15-00:00

Proceedings of the National Academy of Sciences, Vol. 95, No. 9. (28 April 1998), pp. 4959-4963.

10.1073/pnas.95.9.4959

DEVELOPMENT:How Cyanobacteria Count to 10

Robert Haselkorn — 2008-05-08T14:50:39-00:00

Science, Vol. 282, No. 5390. (30 October 1998), pp. 891-892.

10.1126/science.282.5390.891

Life and the Evolution of Earth's Atmosphere

James Kasting — 2008-05-08T07:04:57-00:00

Science, Vol. 296, No. 5570. (10 May 2002), pp. 1066-1068.

10.1126/science.1071184

Predicted Glycosyl Transferase Genes Located outside the HEP Island Are Required for Formation of Heterocyst Envelope Polysaccharide in Anabaena sp. Strain PCC 7120

Yu Wang — 2008-04-28T04:33:51-00:00

J. Bacteriol., Vol. 189, No. 14. (15 July 2007), pp. 5372-5378.

During maturation, heterocysts form an envelope layer of polysaccharide, called heterocyst envelope polysaccharide (HEP), whose synthesis depends on a cluster of genes, the HEP island, and on an additional, distant gene, hepB, or a gene immediately downstream from hepB. We show that HEP formation depends upon the predicted glycosyl transferase genes all4160 at a third locus and alr3699, which is adjacent to hepB and is cotranscribed with it. Mutations in the histidine kinase genes hepN and hepK appear to silence the promoter of hepB and incompletely down-regulate all4160. 10.1128/JB.00343-07

Septum-Localized Protein Required for Filament Integrity and Diazotrophy in the Heterocyst-Forming Cyanobacterium Anabaena sp. Strain PCC 7120

Enrique Flores — 2008-04-28T04:33:04-00:00

J. Bacteriol., Vol. 189, No. 10. (15 May 2007), pp. 3884-3890.

Heterocysts, formed when filamentous cyanobacteria, such as Anabaena sp. strain PCC 7120, are grown in the absence of combined nitrogen, are cells that are specialized in fixing atmospheric nitrogen (N2) under oxic conditions and that transfer fixed nitrogen to the vegetative cells of the filament. Anabaena sp. mutants whose sepJ gene (open reading frame alr2338 of the Anabaena sp. genome) was affected showed filament fragmentation and arrested heterocyst differentiation at an early stage. In a sepJ insertional mutant, a layer similar to a heterocyst polysaccharide layer was formed, but the heterocyst-specific glycolipids were not synthesized. The sepJ mutant did not exhibit nitrogenase activity even when assayed under anoxic conditions. In contrast to proheterocysts produced in the wild type, those produced in the sepJ mutant still divided. SepJ is a multidomain protein whose N-terminal region is predicted to be periplasmic and whose C-terminal domain resembles an export permease. Using a green fluorescent protein translationally fused to the carboxyl terminus of SepJ, we observed that in mature heterocysts and vegetative cells, the protein is localized at the intercellular septa, and when cell division starts, it is localized in a ring whose position is similar to that of a Z ring. SepJ is a novel composite protein needed for filament integrity, proper heterocyst development, and diazotrophic growth. 10.1128/JB.00085-07

Global Gene Expression Patterns of Nostoc punctiforme in Steady-State Dinitrogen-Grown Heterocyst-Containing Cultures and at Single Time Points during the Differentiation of Akinetes and Hormogonia

Elsie Campbell — 2008-04-28T04:30:52-00:00

J. Bacteriol., Vol. 189, No. 14. (15 July 2007), pp. 5247-5256.

The vegetative cells of the filamentous cyanobacterium Nostoc punctiforme can differentiate into three mutually exclusive cell types: nitrogen-fixing heterocysts, spore-like akinetes, and motile hormogomium filaments. A DNA microarray consisting of 6,893 N. punctiforme genes was used to identify the global transcription patterns at single time points in the three developmental states, compared to those in ammonium-grown time zero cultures. Analysis of ammonium-grown cultures yielded a transcriptome of 2,935 genes, which is nearly twice the size of a soluble proteome. The NH4+-grown transcriptome was enriched in genes encoding core metabolic functions. A steady-state N2-grown (heterocyst-containing) culture showed differential transcription of 495 genes, 373 of which were up-regulated. The majority of the up-regulated genes were predicted from studies of heterocyst differentiation and N2 fixation; other genes are candidates for more detailed genetic analysis. Three days into the developmental process, akinetes showed a similar number of differentially expressed genes (497 genes), which were equally up- and down-regulated. The down-regulated genes were enriched in core metabolic functions, consistent with entry into a nongrowth state. There were relatively few adaptive genes up-regulated in 3-day akinetes, and there was little overlap with putative heterocyst developmental genes. There were 1,827 differentially transcribed genes in 24-h hormogonia, which was nearly fivefold greater than the number in akinete-forming or N2-fixing cultures. The majority of the up-regulated adaptive genes were genes encoding proteins for signal transduction and transcriptional regulation, which is characteristic of a motile filament that is poised to sense and respond to the environment. The greatest fraction of the 883 down-regulated genes was involved in core metabolism, also consistent with entry into a nongrowth state. The differentiation of heterocysts (steady state, N2 grown), akinetes, and hormogonia appears to involve the up-regulation of genes distinct for each state. 10.1128/JB.00360-07

A gene cluster that regulates both heterocyst differentiation and pattern formation in Anabaena sp. strain PCC 7120

Wei Zhang — 2008-04-28T04:29:49-00:00

Molecular Microbiology, Vol. 66, No. 6. (2007), pp. 1429-1443.

Summary Wild-type Anabaena sp. strain PCC 7120, a filamentous nitrogen-fixing cyanobacterium, produces single heterocysts at semi-regular intervals. asr0100 (patU5) and alr0101 (patU3) are homologous to the 5' and 3' portions of patU of Nostoc punctiforme. alr0099 (hetZ) overlaps the 5' end of patU5. hetZ, patU5 and patU3 were all upregulated, or expressed specifically, in proheterocysts and heterocysts. Mutants of hetZ showed delayed or no heterocyst differentiation. In contrast, a patU3 mutation produced a multiple contiguous heterocyst (Mch) phenotype and restored the formation of otherwise lost intercalary heterocysts in a patA background. Decreasing the expression of patU3 greatly increased the frequency of heterocysts in a mini-patS strain. Two promoter regions and two principal, corresponding transcripts were detected in the hetZ-patU5-patU3 region. Transcription of hetZ was upregulated in a hetZ mutant and downregulated in a patU3 mutant. When mutants hetZ::C.K2 and hetZ::Tn5-1087b were nitrogen-deprived, PhetC -gfp was very weakly expressed, and in hetZ::Tn5-1087b, PhetR -gfp was relatively strongly expressed in cells that had neither a regular pattern nor altered morphology. We conclude that the hetZ-patU5-patU3 cluster plays an important role in co-ordination of heterocyst differentiation and pattern formation. The presence of homologous clusters in filamentous genera without heterocysts is suggestive of a more general role.

Anabaena sp. strain PCC 7120 gene devH is required for synthesis of the heterocyst glycolipid layer.

ME Ramírez — 2008-04-28T04:28:46-00:00

Journal of bacteriology, Vol. 187, No. 7. (April 2005), pp. 2326-2331.

In response to deprivation for fixed nitrogen, the filamentous cyanobacterium Anabaena sp. strain PCC 7120 provides a microoxic intracellular environment for nitrogen fixation through the differentiation of semiregularly spaced vegetative cells into specialized cells called heterocysts. The devH gene is induced during heterocyst development and encodes a product with characteristics of a trans-acting regulatory protein. A devH mutant forms morphologically distinguishable heterocysts but is Fox(-), incapable of nitrogen fixation in the presence of oxygen. We demonstrate that rearrangements of nitrogen fixation genes take place normally in the devH mutant and that it is Fix(+), i.e., has nitrogenase activity under anoxic conditions. The Fox(-) phenotype was shown by ultrastructural studies to be associated with the absence of the glycolipid layer of the heterocyst envelope. The expression of glycolipid biosynthetic genes in the mutant is greatly reduced, and heterocyst glycolipids are undetectable.

Genes of Cyanobacterial Origin in Plant Nuclear Genomes Point to a Heterocyst-Forming Plastid Ancestor

Oliver Deusch — 2008-04-28T04:28:03-00:00

Mol Biol Evol, Vol. 25, No. 4. (1 April 2008), pp. 748-761.

Plastids are descended from a cyanobacterial symbiosis which occurred over 1.2 billion years ago. During the course of endosymbiosis, most genes were lost from the cyanobacterium's genome and many were relocated to the host nucleus through endosymbiotic gene transfer (EGT). The issue of how many genes were acquired through EGT in different plant lineages is unresolved. Here, we report the genome-wide frequency of gene acquisitions from cyanobacteria in 4 photosynthetic eukaryotes--Arabidopsis, rice, Chlamydomonas, and the red alga Cyanidioschyzon--by comparision of the 83,138 proteins encoded in their genomes with 851,607 proteins encoded in 9 sequenced cyanobacterial genomes, 215 other reference prokaryotic genomes, and 13 reference eukaryotic genomes. The analyses entail 11,569 phylogenies inferred with both maximum likelihood and Neighbor-Joining approaches. Because each phylogenetic result is dependent not only upon the reconstruction method but also upon the site patterns in the underlying alignment, we investigated how the reliability of site pattern generation via alignment affects our results: if the site patterns in an alignment differ depending upon the order in which amino acids are introduced into multiple sequence alignment--N- to C-terminal versus C- to N-terminal--then the phylogenetic result is likely to be artifactual. Excluding unreliable alignments by this means, we obtain a conservative estimate, wherein about 14% of the proteins examined in each plant genome indicate a cyanobacterial origin for the corresponding nuclear gene, with higher proportions (17-25%) observed among the more reliable alignments. The identification of cyanobacterial genes in plant genomes affords access to an important question: From which type of cyanobacterium did the ancestor of plastids arise? Among the 9 cyanobacterial genomes sampled, Nostoc sp. PCC7120 and Anabaena variabilis ATCC29143 were found to harbor collections of genes which are--in terms of presence/absence and sequence similarity--more like those possessed by the plastid ancestor than those of the other 7 cyanobacterial genomes sampled here. This suggests that the ancestor of plastids might have been an organism more similar to filamentous, heterocyst-forming (nitrogen-fixing) representatives of section IV recognized in Stanier's cyanobacterial classification. Members of section IV are very common partners in contemporary symbiotic associations involving endosymbiotic cyanobacteria, which generally provide nitrogen to their host, consistent with suggestions that fixed nitrogen supplied by the endosymbiont might have played an important role during the origin of plastids. 10.1093/molbev/msn022

PrpJ, a PP2C-type protein phosphatase located on the plasma membrane, is involved in heterocyst maturation in the cyanobacterium Anabaena sp. PCC 7120

Jang — 2007-04-22T00:44:30-00:00

Molecular Microbiology, Vol. 64, No. 2. (April 2007), pp. 347-358.

NrrA, a nitrogen-responsive response regulator facilitates heterocyst development in the cyanobacterium Anabaena sp. strain PCC 7120

Shigeki Ehira — 2006-03-09T13:35:33-00:00

Molecular Microbiology, Vol. 59, No. 6. (March 2006), pp. 1692-1703.

From the Cover: CcbP, a calcium-binding protein from Anabaena sp. PCC 7120, provides evidence that calcium ions regulate heterocyst differentiation

Yinhong Zhao — 2008-04-28T04:19:19-00:00

Proceedings of the National Academy of Sciences, Vol. 102, No. 16. (19 April 2005), pp. 5744-5748.

Although it is known that calcium is a very important messenger involved in many eukaryotic cellular processes, much less is known about calcium's role in bacteria. CcbP, a Ca2+-binding protein, was isolated from the heterocystous cyanobacterium Anabaena sp. PCC 7120, and the ccbP gene was cloned and inactivated. In the absence of combined nitrogen, inactivation of ccbP resulted in multiple contiguous heterocysts, whereas overexpression of ccbP suppressed heterocyst formation. Calmodulin, which is not present in Anabaena species, could also suppress heterocyst formation in both Anabaena sp. PCC 7120 and Anabaena variabilis. HetR induction upon nitrogen step-down was slow in the strain overexpressing ccbP. The Ca2+ reporter protein obelin was used to show that mature heterocysts had a high intracellular free Ca2+concentration [Ca2+]i, and immunoblotting showed that CcbP was absent from heterocysts. A regular pattern of cells with higher [Ca2+]i was established during heterocyst differentiation before the appearance of proheterocysts. A rapid increase of [Ca2+]i could be detected 4 h after the removal of combined nitrogen, and this increase was suppressed by excessive CcbP. These results suggest that Ca2+ ions play very important roles in hetR induction and heterocyst differentiation. 10.1073/pnas.0501782102

Mechanism of intercellular molecular exchange in heterocyst-forming cyanobacteria

Conrad Mullineaux — 2008-04-03T16:42:23-00:00

The EMBO Journal, Vol. aop, No. current. (03 April 2008)

From the Cover: Nonmetabolizable analogue of 2-oxoglutarate elicits heterocyst differentiation under repressive conditions in Anabaena sp. PCC 7120

Sophie Laurent — 2008-04-28T04:07:59-00:00

Proceedings of the National Academy of Sciences, Vol. 102, No. 28. (12 July 2005), pp. 9907-9912.

In response to combined nitrogen starvation in the growth medium, the filamentous cyanobacterium Anabaena sp. PCC 7120 is able to develop a particular cell type, called a heterocyst, specialized in molecular nitrogen fixation. Heterocysts are regularly intercalated among vegetative cells and represent 5-10% of all cells along each filament. In unicellular cyanobacteria, the key Krebs cycle intermediate, 2-oxoglutarate (2-OG), has been suggested as a nitrogen status signal, but in vivo evidence is still lacking. In this study we show that nitrogen starvation causes 2-OG to accumulate transiently within cells of Anabaena PCC 7120, reaching a maximal intracellular concentration of approx0.1 mM 1 h after combined nitrogen starvation. A nonmetabolizable fluorinated 2-OG derivative, 2,2-difluoropentanedioic acid (DFPA), was synthesized and used to demonstrate the signaling function of 2-OG in vivo. DFPA is shown to be a structural analogue of 2-OG and the process of its uptake and accumulation in vivo can be followed by 19F magic angle spinning NMR because of the presence of the fluorine atom and its chemical stability. DFPA at a threshold concentration of 0.3 mM triggers heterocyst differentiation under repressing conditions. The multidisciplinary approaches using synthetic fluorinated analogues, magic angle spinning NMR for their analysis in vivo, and techniques of molecular biology provide a powerful means to identify the nature of the signals that remain unknown or poorly defined in many signaling pathways. 10.1073/pnas.0502337102

Regulation of intracellular free calcium concentration during heterocyst differentiation by HetR and NtcA in Anabaena sp. PCC 7120.

Y Shi — 2008-04-28T04:07:38-00:00

Proceedings of the National Academy of Sciences of the United States of America, Vol. 103, No. 30. (25 July 2006), pp. 11334-11339.

Calcium ions are important to some prokaryotic cellular processes, such as heterocyst differentiation of cyanobacteria. Intracellular free Ca(2+)concentration, [Ca(2+)](i), increases several fold in heterocysts and is regulated by CcbP, a Ca(2+)-binding protein found in heterocyst-forming cyanobacteria. We demonstrate here that CcbP is degraded by HetR, a serine-type protease that controls heterocyst differentiation. The degradation depends on Ca(2+) and appears to be specific because HetR did not digest other tested proteins. CcbP was found to bind two Ca(2+) per molecule with K(D) values of 200 nM and 12.8 microM. Degradation of CcbP releases bound Ca(2+) that contributes significantly to the increase of [Ca(2+)](i) during the process of heterocyst differentiation in Anabaena sp. strain PCC 7120. We suggest that degradation of CcbP is a mechanism of positive autoregulation of HetR. The down-regulation of ccbP in differentiating cells and mature heterocysts, which also is critical to the regulation of [Ca(2+)](i), depends on NtcA. Coexpression of ntcA and a ccbP promoter-controlled gfp in Escherichia coli diminished production of GFP, and the decrease is enhanced by alpha-ketoglutarate. It was also found that NtcA could bind a fragment of the ccbP promoter containing an NtcA-binding sequence in a alpha-ketoglutarate-dependent fashion. Therefore, [Ca(2+)](i) is regulated by a collaboration of HetR and NtcA in heterocyst differentiation in Anabaena sp. strain PCC 7120.

Heterocyst differentiation and pattern formation in cyanobacteria: a chorus of signals

Cheng-Cai Zhang — 2005-12-23T13:10:05-00:00

Molecular Microbiology, Vol. 59, No. 2. (January 2006), pp. 367-375.

Characterization of a gene controlling heterocyst differentiation in the cyanobacterium Anabaena 7120.

WJ Buikema — 2008-04-27T23:02:54-00:00

Genes Dev., Vol. 5, No. 2. (1 February 1991), pp. 321-330.

Anabaena 7120 mutant 216 fails to differentiate heterocyst. We previously identified a 2.4-kb wild-type DNA fragment able to complement this mutant. We show here that the sequence of this fragment contains a single open reading frame (hetR), encoding a 299-amino-acid protein. Conjugation of deletion subclones of this fragment into strain 216 showed that the hetR-coding region is both necessary and sufficient for complementation of the Het- phenotype. The mutation in 216 is located at nucleotide 535 in the hetR gene, converting a serine at position 179 in the wild-type protein to an asparagine in the mutant. Interruption of the hetR gene in wild-type cells results in a mutant phenotype identical to that of 216. Both 216 and wild-type cells containing wild-type hetR on a plasmid display increased frequency of heterocysts, even on media containing fixed nitrogen. These results suggest that hetR encodes a product that is not only essential for but also controls heterocyst development. This putative regulatory protein lacks known structural motifs characteristic of transcription factors and probably acts at a level one or more steps removed from its target genes. 10.1101/gad.5.2.321

Anabaena sp. strain PCC 7120 ntcA gene required for growth on nitrate and heterocyst development.

TF Wei — 2008-04-17T00:30:28-00:00

J. Bacteriol., Vol. 176, No. 15. (1 August 1994), pp. 4473-4482.

The Anabaena sp. strain PCC 7120 ntcA (bifA) gene encodes a sequence-specific DNA-binding protein, NtcA (BifA, VF1) that interacts with the upstream region of several genes, including glnA, xisA, rbcL, and nifH. We have constructed a ntcA null mutant by interrupting the gene with an omega Spr-Smr cassette. The ntcA mutant was not able to grow with nitrate or atmospheric dinitrogen as the sole nitrogen source but could be grown on medium containing ammonium. The ntcA mutant was unable to form heterocysts and did not rearrange the nifD or fdxN elements after induction on a medium lacking combined nitrogen. Northern (RNA) analysis of ntcA in the wild-type strain during nitrogen stepdown showed a peak of ntcA message at an early stage (12 h) of heterocyst induction. Complementation of the ntcA mutant with a DNA fragment containing the ntcA gene and 251 bp of upstream sequence on a shuttle vector restored a wild-type phenotype; however, a similar construction containing 87 bp of upstream sequence only partially restored the phenotype. Northern analysis of RNA samples isolated from ammonium-grown cultures of the ntcA mutant showed reduced amounts of glnA message and the absence of a 1.7-kb transcript. In the wild type, the 1.7-kb transcript represents the majority of glnA transcripts after nitrogen stepdown. The ntcA mutant showed a normal pattern of rbcLS messages under these growth conditions.

Synthesis of nitrogenase in mutants of the cyanobacterium Anabaena sp. strain PCC 7120 affected in heterocyst development or metabolism.

A Ernst — 2008-04-17T00:28:38-00:00

Journal of bacteriology, Vol. 174, No. 19. (October 1992), pp. 6025-6032.

Mutants of Anabaena sp. strain PCC 7120 that are incapable of sustained growth with air as the sole source of nitrogen were generated by using Tn5-derived transposons. Nitrogenase was expressed only in mutants that showed obvious morphological signs of heterocyst differentiation. Even under rigorously anaerobic conditions, nitrogenase was not synthesized in filaments that were unable to develop heterocysts. These results suggest that competence to synthesize nitrogenase requires a process that leads to an early stage of visible heterocyst development and are consistent with the idea that synthesis of nitrogenase is under developmental control (J. Elhai and C. P. Wolk, EMBO J. 9:3379-3388, 1990). We isolated mutants in which differentiation was arrested at an intermediate stage of heterocyst formation, suggesting that differentiation proceeds in stages; those mutants, as well as mutants with aberrant heterocyst envelopes and a mutant with defective respiration, expressed active nitrogenase under anaerobic conditions only. These results support the idea that the heterocyst envelope and heterocyst respiration are required for protection of nitrogenase from inactivation by oxygen. In the presence of air, such mutants contained less nitrogenase than under anaerobic conditions, and the Fe-protein was present in a posttranslationally modified inactive form. We conclude that internal partial oxygen pressure sufficient to inactivate nitrogenase is insufficient to repress synthesis of the enzyme completely. Among mutants with an apparently intact heterocyst envelope and normal respiration, three had virtually undetectable levels of dinitrogenase reductase under all conditions employed. However, three others expressed oxygen-sensitive nitrogenase activity, suggesting that respiration and barrier to diffusion of gases may not suffice for oxygen protection of nitrogenase in these mutants; two of these mutants reduced acetylene to ethylene and ethane.

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

TA Black — 2008-04-17T00:27:39-00:00

Journal of bacteriology, Vol. 176, No. 8. (April 1994), pp. 2282-2292.

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.

PatS and Products of Nitrogen Fixation Control Heterocyst Pattern

Ho-Sung Yoon — 2008-04-17T00:26:57-00:00

J. Bacteriol., Vol. 183, No. 8. (15 April 2001), pp. 2605-2613.

The filamentous cyanobacterium Anabaena sp. strain PCC 7120 forms a developmental pattern of single heterocysts separated by approximately 10 vegetative cells. Heterocysts differentiate from vegetative cells and are specialized for nitrogen fixation. The patS gene, which encodes a small peptide that inhibits heterocyst differentiation, is expressed in proheterocysts and plays a critical role in establishing the heterocyst pattern. Here we present further analysis of patS expression and heterocyst pattern formation. A patS-gfp reporter strain revealed clusters of patS-expressing cells during the early stage of heterocyst differentiation. PatS signaling is likely to be involved in the resolution of these clusters. Differentiating cells were inhibited by PatS during the time period 6 to 12 h after heterocyst induction, when groups of differentiating cells were being resolved to a single proheterocyst. Increased transcription of patS during development coincided with expression from a new transcription start site. In vegetative cells grown on nitrate, the 5' end of a transcript for patS was localized 314 bases upstream from the first translation initiation codon. After heterocyst induction, a new transcript with a 5' end at [-]39 bases replaced the vegetative cell transcript. A patS mutant grown for several days under nitrogen-fixing conditions showed partial restoration of the normal heterocyst pattern, presumably because of a gradient of nitrogen compounds supplied by the heterocysts. The patS mutant formed heterocysts when grown in the presence of nitrate but showed no nitrogenase activity and no obvious heterocyst pattern. We conclude that PatS and products of nitrogen fixation are the main signals determining the heterocyst pattern. 10.1128/JB.183.8.2605-2613.2001

Expression of the Anabaena hetR gene from a copper-regulated promoter leads to heterocyst differentiation under repressing conditions

William Buikema — 2008-04-17T00:25:47-00:00

Proceedings of the National Academy of Sciences, Vol. 98, No. 5. (27 February 2001), pp. 2729-2734.

10.1073/pnas.051624898

The hetC Gene Is a Direct Target of the NtcA Transcriptional Regulator in Cyanobacterial Heterocyst Development

Alicia Muro-Pastor — 2008-04-17T00:24:44-00:00

J. Bacteriol., Vol. 181, No. 21. (1 November 1999), pp. 6664-6669.

The heterocyst is the site of nitrogen fixation in aerobically grown cultures of some filamentous cyanobacteria. Heterocyst development in Anabaena sp. strain PCC 7120 is dependent on the global nitrogen regulator NtcA and requires, among others, the products of the hetR and hetC genes. Expression of hetC, tested by RNA- DNA hybridization, was impaired in an ntcA mutant. A nitrogen-regulated, NtcA-dependent putative transcription start point was localized at nucleotide [-]571 with respect to the hetC translational start. Sequences upstream from this transcription start point exhibit the structure of the canonical cyanobacterial promoter activated by NtcA, and purified NtcA protein specifically bound to a DNA fragment containing this promoter. Activation of expression of hetC during heterocyst development appears thus to be directly operated by NtcA. NtcA-mediated activation of hetR expression was not impaired in a hetC mutant, indicating that HetC is not an NtcA-dependent element required for hetR induction.

Independent regulation of nifHDK operon transcription and DNA rearrangement during heterocyst differentiation in the cyanobacterium Anabaena sp. strain PCC 7120.

JW Golden — 2008-04-17T00:22:53-00:00

Journal of bacteriology, Vol. 173, No. 22. (November 1991), pp. 7098-7105.

The filamentous cyanobacterium Anabaena sp. strain PCC 7120 expresses the genes required for nitrogen fixation in terminally differentiated cells called heterocysts. The nifHDK operon encodes the nitrogenase polypeptides and is expressed at high levels in heterocysts. During heterocyst differentiation, an 11-kb DNA element is excised from the nifD gene by site-specific recombination. The xisA gene, located on the 11-kb element, is required for the excision of the element. Transcription and DNA rearrangement of the nifHDK operon both occur late during heterocyst differentiation, about 18 to 24 h after induction, suggesting that the regulation of these events might be coupled. We show that heterocyst-specific transcription and DNA rearrangement of the nifHDK operon are independent of one another. Northern (RNA) analysis of the xisA mutant strain DW12-2.2, which cannot excise the nifD 11-kb element or fix nitrogen, showed that the nifH and nifD genes are transcribed on unrearranged chromosomes. The nifK gene was not transcribed in DW12-2.2, indicating that its expression is dependent on the nifH promoter and excision of the 11-kb element from the operon. A 1.68-kb DNA fragment containing the nifH promoter was deleted from the chromosome to produce the mutant strain LW1. LW1 formed heterocysts but did not grow on nitrogen-free medium and showed no transcription through nifD. Southern analysis of LW1 showed normal excision of the 11-kb element from the nifHDK operon, indicating that transcription from the nifH promoter is not required for the developmentally regulated DNA rearrangement.

The hglK gene is required for localization of heterocyst-specific glycolipids in the cyanobacterium Anabaena sp. strain PCC 7120.

K Black — 2008-04-17T00:20:18-00:00

Journal of bacteriology, Vol. 177, No. 22. (November 1995), pp. 6440-6448.

Mutant strain 543 of the cyanobacterium Anabaena sp. strain PCC 7120 was originally isolated as a Fox- mutant following chemical mutagenesis. Ultrastructural analysis shows that in nitrogen-replete media the vegetative cells of the mutant are more cylindrical and have thicker septa than those of the wild type, while in nitrogen-free media the mutant heterocysts lack the normal glycolipid layer external to the cell wall. Although this layer is absent, strain 543 heterocysts nevertheless contain heterocyst-specific glycolipids, as determined by thin-layer chromatography. The mutation in strain 543 is in a gene we have named hglK, encoding a protein of 727 amino acids. The wild-type HglK protein appears to contain four membrane-spanning regions followed by 36 repeats of a degenerate pentapeptide sequence, AXLXX. The mutation in strain 543 introduces a termination codon immediately upstream of the pentapeptide repeat region. A mutant constructed by insertion of an antibiotic resistance cassette near the beginning of the hglK gene has the same phenotype as strain 543. We propose that hglK encodes a protein necessary for the localization of heterocyst glycolipids and that this function requires the pentapeptide repeats of the HglK protein.

Is the Heterocyst the Site of Nitrogen Fixation in Blue-green Algae?

P Fay — 2008-04-17T00:19:37-00:00

Nature, Vol. 220, No. 5169. (23 November 1968), pp. 810-812.

Deletion of a 55-kilobase-pair DNA element from the chromosome during heterocyst differentiation of Anabaena sp. strain PCC 7120.

JW Golden — 2008-04-17T00:18:41-00:00

Journal of bacteriology, Vol. 170, No. 11. (November 1988), pp. 5034-5041.

The filamentous cyanobacterium Anabaena sp. strain PCC 7120 produces terminally differentiated heterocysts in response to a lack of combined nitrogen. Heterocysts are found approximately every 10th cell along the filament and are morphologically and biochemically specialized for nitrogen fixation. At least two DNA rearrangements occur during heterocyst differentiation in Anabaena sp. strain PCC 7120, both the result of developmentally regulated site-specific recombination. The first is an 11-kilobase-pair (kb) deletion from within the 3' end of the nifD gene. The second rearrangement occurs near the nifS gene but has not been completely characterized. The DNA sequences found at the recombination sites for each of the two rearrangements show no similarity to each other. To determine the topology of the rearrangement near the nifS gene, cosmid libraries of vegetative-cell genomic DNA were constructed and used to clone the region of the chromosome involved in the rearrangement. Cosmid clones which spanned the DNA separating the two recombination sites that define the ends of the element were obtained. The restriction map of this region of the chromosome showed that the rearrangement was the deletion of a 55-kb DNA element from the heterocyst chromosome. The excised DNA was neither degraded nor amplified, and its function, if any, is unknown. The 55-kb element was not detectably transcribed in either vegetative cells or heterocysts. The deletion resulted in placement of the rbcLS operon about 10 kb from the nifS gene on the chromosome. Although the nifD 11-kb and nifS 55-kb rearrangements both occurred under normal aerobic heterocyst-inducing conditions, only the 55-kb excision occurred in argon-bubbled cultures, indicating that the two DNA rearrangements can be regulated differently.

The patA gene product, which contains a region similar to CheY of Escherichia coli, controls heterocyst pattern formation in the cyanobacterium Anabaena 7120.

J Liang — 2008-04-17T00:18:00-00:00

Proceedings of the National Academy of Sciences of the United States of America, Vol. 89, No. 12. (15 June 1992), pp. 5655-5659.

In Anabaena 7120, heterocysts (cells specialized for nitrogen fixation) develop at the ends of filaments and at intervals within each filament. We have isolated a mutant Anabaena strain that develops heterocysts mostly at the ends of filaments. This mutant, PAT-1, grows poorly under nitrogen-fixing conditions. The wild-type gene that complements the mutation in PAT-1, called patA, was cloned and sequenced. The predicted PatA protein contains 379 amino acids distributed among three "domains" based on predictions of hydropathy and flexibility. The carboxyl-terminal domain is very similar to that of CheY and other response regulators in two-component regulatory systems in eubacteria. The patA mutation suppresses the multiheterocyst phenotype produced by extra copies of the wild-type hetR gene described previously, suggesting that PatA and HetR are components of the same environment-sensing regulatory circuit in Anabaena.

Two Anabaena sp. strain PCC 7120 DNA-binding factors interact with vegetative cell- and heterocyst-specific genes.

TS Ramasubramanian — 2008-04-17T00:17:17-00:00

Journal of bacteriology, Vol. 176, No. 5. (March 1994), pp. 1214-1223.

The DNA-binding factor BifA (previously called VF1) binds upstream of the developmentally regulated site-specific recombinase gene xisA in the cyanobacterium Anabaena sp. strain PCC 7120. Besides binding xisA, BifA also binds the glnA, rbcL, and nifH promoter regions. DNase I footprint analysis of BifA binding to glnA showed a protected region -125 to -148 bp upstream of the translation start site. The binding site is between the major glnA transcription start site used in vegetative cells (RNAII) and the major transcription start site used under nitrogen-deficient conditions (RNAI). The two BifA-binding sites on the rbcL promoter were localized to a 24-bp region from +12 to -12 nucleotides and to a 12-bp region from -43 to -54 nucleotides with respect to the transcription start site. Comparison of the BifA binding sites on the glnA, xisA, and rbcL upstream regions revealed the consensus recognition sequence TGT(N9 or 10) ACA. We have identified a second DNA-binding activity (factor 2) that interacts with rbcL and xisA upstream regions. Factor 2 can be resolved from BifA by heparin-Sepharose chromatography and was present in a bifA mutant. Analysis of partially purified vegetative cell and heterocyst extracts showed that whereas BifA was present in both cell types, factor 2 was present only in vegetative cells. DNase I footprint analysis of factor 2 binding to rbcL showed protection of a 63-bp region between positions -15 and -77 with respect to the transcription start site. The factor 2 binding site on xisA was localized to a 68-bp region that showed considerable overlap with the BifA binding sites.

HETEROCYST FORMATION

Peter Wolk — 2008-04-17T00:14:39-00:00

Annual Review of Genetics, Vol. 30, No. 1. (1996), pp. 59-78.

Abstract Heterocysts are microaerobic, N2-fixing cells that form in a patterned array within O2-producing filamentous cyanobacteria. Structural features of heterocysts can be predicted from consideration of their physiology. This review focuses on the spacing mechanism that determines which cells will differentiate, and on the regulation of the progression of the differentiation process. Applicable genetic tools, developed primarily using Anabaena PCC 7120, but employed also with Nostoc spp., are reviewed. These tools include localization of transcription using fusions to lux, lac, and gfp, and mutagenesis with oriV-containing derivatives of transposon Tn5. Mature and developing heterocysts inhibit nearby vegetative cells from differentiating; genes patA, devA, hetC, and the hetMNI locus may hold keys to understanding intercellular interactions that influence heterocyst formation. Regulatory and other genes that are transcriptionally activated at different times after nitrogen stepdown have been identified, and should permit analysis of mechanisms that underlie the progression of heterocyst differentiation.