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James W. Golden - One of the best experts on this subject based on the ideXlab platform.
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Heterocyst Development and Pattern Formation
Chemical Communication among Bacteria, 2014Co-Authors: M. Ramona Aldea, Krithika Kumar, James W. GoldenAbstract:Most of the information about Heterocyst development to date is based on the study of three species of Heterocyst-forming filamentous cyanobacteria: Anabaena (also Nostoc) sp. strain PCC 7120, A. variabilis ATCC 29413, and Nostoc punctiforme ATCC 29133. This chapter focuses on those genes involved in signaling and regulation. HetR plays a central role in Heterocyst development and pattern formation. In bacteria, calcium ions play important roles in various cellular processes such as pathogenesis, sporulation in Bacillus, chemotaxis in Escherichia coli, and Heterocyst development in cyanobacteria. PatA may influence Heterocyst development by attenuating the negative effects of the main inhibitory signals of Heterocyst pattern formation, PatS and HetN. Late stages of Heterocyst development are characterized by structural changes that include the deposition of three cell layers: an outermost fibrous layer, an envelope polysaccharide layer, and an innermost glycolipid layer. During nitrogen fixation, nitrogenase reduces atmospheric nitrogen to ammonia, which is then assimilated into amino acids. A recent epistasis analysis of four genes involved in pattern formation in Anabaena Strain PCC 7120 suggests that PatA has two distinct activities, to promote differentiation as well as to attenuate the negative effects of PatS and HetN on differentiation. Some genes that are required for Heterocyst development are also involved in akinete formation, such as hetR and hepA. In the absence of Heterocysts, the akinetes seem to form at random positions along the filament, whereas the presence of Heterocysts influences akinete positioning, implying the existence of cell-to-cell communication.
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overexpression of pkne blocks Heterocyst development in anabaena sp strain pcc 7120
Journal of Bacteriology, 2011Co-Authors: Sushanta Kumar Saha, James W. GoldenAbstract: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. PpknE-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. The filamentous cyanobacterium Anabaena (Nostoc) sp. strain PCC 7120 (here called Anabaena) is capable of fixing atmospheric nitrogen in specialized cells called Heterocysts. During growth on nitrate- or ammonium-containing medium, Anabaena filaments possess only photosynthetic vegetative cells. When combined nitrogen becomes insufficient in the growth medium, approximately 10% of the vegetative cells differentiate into Heterocysts semiregularly spaced along each filament. Genome-wide expression analysis revealed global changes in gene expression in Anabaena filaments upon nitro
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the sige gene is required for normal expression of Heterocyst specific genes in anabaena sp strain pcc 7120
Journal of Bacteriology, 2011Co-Authors: Krithika Kumar, Rodrigo A Mellaherrera, M R Neunuebel, Sushanta Kumar Saha, James W. GoldenAbstract:ABSTRACT The filamentous cyanobacterium Anabaena (Nostoc) sp. strain PCC 7120 produces specialized cells for nitrogen fixation called Heterocysts. Previous work showed that the group 2 sigma factor sigE (alr4249; previously called sigF) is upregulated in differentiating Heterocysts 16 h after nitrogen step-down. We now show that the sigE gene is required for normal Heterocyst development and normal expression levels of several Heterocyst-specific genes. Mobility shift assays showed that the transcription factor NtcA binds to sites in the upstream region of sigE and that this binding is enhanced by 2-oxoglutarate (2-OG). Deletions of the region containing the NtcA binding sites in PsigE-gfp reporter plasmids showed that the sites contribute to normal developmental regulation but are not essential for upregulation in Heterocysts. Northern RNA blot analysis of nifH mRNA revealed delayed and reduced transcript levels during Heterocyst differentiation in a sigE mutant background. Quantitative reverse transcription-PCR (qRT-PCR) analyses of the sigE mutant showed lower levels of transcripts for nifH, fdxH, and hglE2 but normal levels for hupL. We developed a PnifHD-gfp reporter construct that showed strong Heterocyst-specific expression. Time-lapse microscopy of the PnifHD-gfp reporter in a sigE mutant background showed delayed development and undetectable green fluorescent protein (GFP) fluorescence. Overexpression of sigE caused accelerated Heterocyst development, an increased Heterocyst frequency, and premature expression of GFP fluorescence from the PnifHD-gfp reporter.
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Cyanobacterial Heterocysts.
Cold Spring Harbor perspectives in biology, 2010Co-Authors: Krithika Kumar, Rodrigo A Mella-herrera, James W. GoldenAbstract:Many multicellular cyanobacteria produce specialized nitrogen-fixing Heterocysts. During diazotrophic growth of the model organism Anabaena (Nostoc) sp. strain PCC 7120, a regulated developmental pattern of single Heterocysts separated by about 10 to 20 photosynthetic vegetative cells is maintained along filaments. Heterocyst structure and metabolic activity function together to accommodate the oxygen-sensitive process of nitrogen fixation. This article focuses on recent research on Heterocyst development, including morphogenesis, transport of molecules between cells in a filament, differential gene expression, and pattern formation.
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the anabaena sp strain pcc 7120 gene all2874 encodes a diguanylate cyclase and is required for normal Heterocyst development under high light growth conditions
Journal of Bacteriology, 2008Co-Authors: Ramona M Neunuebel, James W. GoldenAbstract:The genome of the Heterocyst-forming cyanobacterium Anabaena sp. strain PCC 7120 harbors 14 genes containing a GGDEF diguanylate cyclase domain. We found that inactivation of one of these genes, all2874, caused abnormal Heterocyst development. The all2874 mutant showed a pronounced reduction in Heterocyst frequency during diazotrophic growth and reduced vegetative cell size compared to the wild type. The severity of the mutant phenotype varied with light intensity; at high light intensity, the mutant phenotype was accentuated, whereas at low light intensity the phenotype was similar to wild type. Under high-light growth conditions, the initial Heterocyst frequency and pattern for the all2874 mutant were normal, but within 4 days following nitrogen step-down, many intervals between Heterocysts increased to as many as 200 vegetative cells, whereas in the wild type the intervals were less than 25 vegetative cells. Filaments containing these unusually long vegetative cell intervals between Heterocysts also contained intervals of normal length. An all2874 mutant strain carrying a P(patS)-gfp transcriptional reporter fusion failed to show normal upregulation of the reporter, which indicates that the decrease in Heterocyst frequency is due to an early block in differentiation before induction of the patS gene, which in the wild type takes place 8 h after nitrogen step-down. Genetic epistasis experiments suggest that All2874 acts upstream of the master regulator HetR in differentiating cells. We also showed that purified All2874 functions as a diguanylate cyclase in vitro. We hypothesize that All2874 is required for the normal regulation of Heterocyst frequency under high-light growth conditions.
Enrique Flores - One of the best experts on this subject based on the ideXlab platform.
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Heterocyst septa contain large nanopores that are influenced by the fra proteins in the filamentous cyanobacterium anabaena
Journal of Bacteriology, 2021Co-Authors: Sergio Arevalo, Enrique FloresAbstract:Multicellular Heterocyst-forming cyanobacteria such as Anabaena grow as chains of cells forming filaments that, under diazotrophic conditions, contain two cell types: vegetative cells that perform oxygenic photosynthesis and N2-fixing Heterocysts. Along the filament, the intercellular septa contain a thick peptidoglycan layer that forms septal disks. Proteinaceous septal junctions connect the cells in the filament traversing the septal disks through nanopores. The fraCDE operon encodes proteins needed to make long filaments in Anabaena FraC and FraD, located at the intercellular septa, are involved in the formation of septal junctions. Using a superfolder-GFP fusion, here we show that FraE is mainly localized to the poles of the Heterocysts, consistent with the requirement of FraE for constriction of the Heterocyst poles to form the "Heterocyst neck". A fraE insertional mutant was impaired by 22% to 38% in transfer of fluorescent calcein from vegetative cells to Heterocysts. Septal disks were inspected in murein sacculi from Heterocyst-enriched preparations. Unexpectedly, the diameter of the nanopores in Heterocyst septa was about 1.5- to 2-fold larger than in vegetative cell septa. The number of these nanopores was 76% and 6% of the wild-type number in fraE or a fraC fraD mutant, respectively. Our results show that FraE is mainly involved in Heterocyst maturation whereas FraC and FraD are needed for the formation of the large nanopores of Heterocyst septa as they are for vegetative cell nanopores. Additionally, arrays of small pores conceivably involved in polysaccharide export were observed close to the disks in the Heterocyst murein sacculi preparations.IMPORTANCEIntercellular communication, an essential attribute of multicellularity, is required for diazotrophic growth in Heterocyst-forming cyanobacteria such as Anabaena, in which the cells are connected by proteinaceous septal junctions that are structural analogs of metazoan connexons. The septal junctions allow molecular intercellular diffusion traversing the septal peptidoglycan through nanopores. In Anabaena the fraCDE operon encodes septal proteins essential for intercellular communication. FraC and FraD are components of the septal junctions along the filament, whereas here we show that FraE is mainly present at the Heterocyst poles. We found that the intercellular septa in murein sacculi from Heterocysts contain nanopores that are larger than those in vegetative cells, establishing a previously unknown difference between Heterocyst and vegetative cell septa in Anabaena.
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Developmental Biology in Cyanobacteria
MDPI AG, 2019Co-Authors: Antonia Herrero, Enrique FloresAbstract:Filamentous, Heterocyst-forming cyanobacteria are phototrophic multicellular organisms in which N2-fixing Heterocysts and CO2-fixing vegetative cells exchange regulators and nutrients [...
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Homospermidine biosynthesis in the cyanobacterium Anabaena requires a deoxyhypusine synthase homologue and is essential for normal diazotrophic growth.
Molecular microbiology, 2018Co-Authors: Mireia Burnat, Sok Ho Kim, Anthony J. Michael, Enrique FloresAbstract:Polyamines are primordial, small organic polycations present in almost all cells, but their roles in bacteria are poorly understood. sym-Homospermidine is the dominant polyamine in the filamentous, N2 -fixing, Heterocyst-forming cyanobacterium Anabaena sp. PCC 7120. Synthesis of homospermidine was dependent on speA (encoding arginine decarboxylase), speB (agmatinase) and speY (deoxyhypusine synthase homologue), which in bacteria is an unprecedented pathway. Inactivation of any of these genes impaired diazotrophic growth. Heterocyst differentiation in the speA mutant was blocked at an early step, after induction of the regulatory gene hetR but before production of Heterocyst-specific glycolipids (HGL). In contrast, the speY mutant produced HGL and showed slow diazotrophic growth. Analysis of fusions to green fluorescent protein revealed that SpeA (like SpeB previously described) accumulates at higher levels in vegetative cells than in Heterocysts, and that SpeY accumulates in vegetative cells but also at significant levels in Heterocysts. The homospermidine biosynthetic pathway is therefore active primarily in vegetative cells but the last step can be completed in Heterocysts. Our findings indicate an important role for polyamines in the diazotrophic biology of Anabaena. Furthermore, inactivation of a gene cluster (potADB) encoding a polyamine ABC transporter disrupted diazotrophic growth, corroborating the importance of polyamine homeostasis in Anabaena.
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overexpression of sepj alters septal morphology and Heterocyst pattern regulated by diffusible signals in anabaena
Molecular Microbiology, 2016Co-Authors: Vicente Mariscal, Antonia Herrero, Conrad W. Mullineaux, Dennis J Nurnberg, Enrique FloresAbstract: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.
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relationships between the abc exporter hetc and peptides that regulate the spatiotemporal pattern of Heterocyst distribution in anabaena
PLOS ONE, 2014Co-Authors: Laura Corralesguerrero, Enrique Flores, Antonia HerreroAbstract:In the model cyanobacterium Anabaena sp. PCC 7120, cells called Heterocysts that are specialized in the fixation of atmospheric nitrogen differentiate from vegetative cells of the filament in the absence of combined nitrogen. Heterocysts follow a specific distribution pattern along the filament, and a number of regulators have been identified that influence the Heterocyst pattern. PatS and HetN, expressed in the differentiating cells, inhibit the differentiation of neighboring cells. At least PatS appears to be processed and transferred from cell to cell. HetC is similar to ABC exporters and is required for differentiation. We present an epistasis analysis of these regulatory genes and of genes, hetP and asr2819, successively downstream from hetC, and we have studied the localization of HetC and HetP by use of GFP fusions. Inactivation of patS, but not of hetN, allowed differentiation to proceed in a hetC background, whereas inactivation of hetC in patS or patS hetN backgrounds decreased the frequency of contiguous proHeterocysts. A HetC-GFP protein is localized to the Heterocysts and especially near their cell poles, and a putative HetC peptidase domain was required for Heterocyst differentiation but not for HetC-GFP localization. hetP is also required for Heterocyst differentiation. A HetP-GFP protein localized mostly near the Heterocyst poles. ORF asr2819, which we denote patC, encodes an 84-residue peptide and is induced upon nitrogen step-down. Inactivation of patC led to a late spreading of the Heterocyst pattern. Whereas HetC and HetP appear to have linked functions that allow Heterocyst differentiation to progress, PatC may have a role in selecting sites of differentiation, suggesting that these closely positioned genes may be functionally related.
Chengcai Zhang - One of the best experts on this subject based on the ideXlab platform.
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patd a gene regulated by ntca is involved in the optimization of Heterocyst frequency in the cyanobacterium anabaena sp strain pcc 7120
Journal of Bacteriology, 2019Co-Authors: Li Wang, J Zhang, Wenli Chen, Guiming Lin, Tiancai Niu, Shaoran Zhang, Guofang Tang, Chengcai ZhangAbstract:In the filamentous multicellular cyanobacterium Anabaena sp. strain PCC 7120, 5 to 10% of the cells differentiate into Heterocysts, which are specialized in N2 fixation. Heterocysts and vegetative cells are mutually dependent for filament growth through nutrient exchange. Thus, the Heterocyst frequency should be optimized to maintain the cellular carbon and nitrogen (C/N) balance for filament fitness in the environment. Here, we report the identification of patD, whose expression is directly activated in developing cells by the transcription factor NtcA. The inactivation of patD increases Heterocyst frequency and promotes the upregulation of the positive regulator of Heterocyst development hetR, whereas its overexpression decreases the Heterocyst frequency. The change in Heterocyst frequency resulting from the inactivation of patD leads to the reduction in competitiveness of the filaments under combined-nitrogen-depleted conditions. These results indicate that patD regulates Heterocyst frequency in Anabaena sp. PCC 7120, ensuring its optimal filament growth.IMPORTANCE Microorganisms have evolved various strategies in order to adapt to the environment and compete with other organisms. Heterocyst differentiation is a prokaryotic model for studying complex cellular regulation. The NtcA-regulated gene patD controls the ratio of Heterocysts relative to vegetative cells on the filaments of Anabaena sp. strain PCC 7120. Such a regulation provides a mechanism through which carbon fixation by vegetative cells and nitrogen fixation by Heterocysts are properly balanced to ensure optimal growth and keep a competitive edge for long-term survival.
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Dynamics and Cell-Type Specificity of the DNA Double-Strand Break Repair Protein RecN in the Developmental Cyanobacterium Anabaena sp. Strain PCC 7120
PLoS ONE, 2015Co-Authors: Jinglan Wang, Li Wang, Chengcai Zhang, Wenli ChenAbstract: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 filamen-tous cyanobacterium Anabaena sp. strain PCC 7120 is capable of forming Heterocysts for N 2 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.
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hetr and pats two genes necessary for Heterocyst pattern formation are widespread in filamentous nonHeterocyst forming cyanobacteria
Microbiology, 2009Co-Authors: J Zhang, Wenli Chen, Chengcai ZhangAbstract:Heterocysts, cells specialized in N2 fixation in cyanobacteria, appeared at near to 2.1 Ga. They constitute one of the oldest forms of differentiated cells in evolution, and are thus an interesting model for studies on evolutionary-developmental biology. How Heterocysts arose during evolution remains unknown. In Anabaena PCC 7120, Heterocyst development requires, among other genes, hetR for the initiation of Heterocyst differentiation, and patS, encoding a diffusible inhibitor of Heterocyst formation. In this study, we report that both hetR and patS are widespread among filamentous cyanobacteria that do not form Heterocysts or fix N2. hetR and patS are found in proximity on the chromosome in several cases, such as Arthrospira platensis, in which the level of HetR increased following nitrogen deprivation. The hetR gene of A. platensis could complement a hetR mutant of Anabaena PCC 7120, and patS of A. platensis could suppress Heterocyst differentiation in Anabaena PCC 7120. Thus, key regulatory genes, including hetR and patS, involved in Heterocyst development may have evolved before Heterocysts appeared, suggesting that their function was not limited to Heterocyst differentiation.
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prpj a pp2c type protein phosphatase located on the plasma membrane is involved in Heterocyst maturation in the cyanobacterium anabaena sp pcc 7120
Molecular Microbiology, 2007Co-Authors: Jichan Jang, Chengcai Zhang, Li Wang, Robert JeanjeanAbstract:Summary Protein phosphatases play important roles in the regulation of cell growth, division and differentiation. The cyanobacterium Anabaena PCC 7120 is able to differentiate Heterocysts specialized in nitrogen fixation. To protect the nitrogenase from inactivation by oxygen, Heterocyst envelope possesses a layer of polysaccharide and a layer of glycolipids. In the present study, we characterized All1731 (PrpJ), a protein phosphatase from Anabaena PCC 7120. prpJ was constitutively expressed in both vegetative cells and Heterocysts. Under diazotrophic conditions, the mutant ΔprpJ (S20) did not grow, lacked only one of the two Heterocyst glycolipids, and fragmented extensively at the junctions between developing cells and vegetative cells. No Heterocyst glycolipid layer could be observed in the mutant by electron microscopy. The inactivation of prpJ affected the expression of hglEA and nifH, two genes necessary for the formation of the glycolipid layer of Heterocysts and the nitrogenase respectively. PrpJ displayed a phosphatase activity characteristic of PP2C-type protein phosphatases, and was localized on the plasma membrane. The function of prpJ establishes a new control point for Heterocyst maturation because it regulates the synthesis of only one of the two Heterocyst glycolipids while all other genes so far analysed regulate the synthesis of both Heterocyst glycolipids.
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inhibition of cell division suppresses Heterocyst development in anabaena sp strain pcc 7120
Journal of Bacteriology, 2006Co-Authors: Samer Sakr, Chengcai Zhang, Robert Jeanjean, Tania ArcondeguyAbstract:When the filamentous cyanobacterium Anabaena PCC 7120 is exposed to combined nitrogen starvation, 5 to 10% of the cells along each filament at semiregular intervals differentiate into Heterocysts specialized in nitrogen fixation. Heterocysts are terminally differentiated cells in which the major cell division protein FtsZ is undetectable. In this report, we provide molecular evidence indicating that cell division is necessary for Heterocyst development. FtsZ, which is translationally fused to the green fluorescent protein (GFP) as a reporter, is found to form a ring structure at the mid-cell position. SulA from Escherichia coli inhibits the GTPase activity of FtsZ in vitro and prevents the formation of FtsZ rings when expressed in Anabaena PCC 7120. The expression of sulA arrests cell division and suppresses Heterocyst differentiation completely. The antibiotic aztreonam, which is targeted to the FtsI protein necessary for septum formation, has similar effects on both cell division and Heterocyst differentiation, although in this case, the FtsZ ring is still formed. Therefore, Heterocyst differentiation is coupled to cell division but independent of the formation of the FtsZ ring. Consistently, once the inhibitory pressure of cell division is removed, cell division should take place first before Heterocyst differentiation resumes at a normal frequency. The arrest of cell division does not affect the accumulation of 2-oxoglutarate, which triggers Heterocyst differentiation. Consistently, a nonmetabolizable analogue of 2-oxoglutarate does not rescue the failure of Heterocyst differentiation when cell division is blocked. These results suggest that the control of Heterocyst differentiation by cell division is independent of the 2-oxoglutarate signal.
Antonia Herrero - One of the best experts on this subject based on the ideXlab platform.
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Developmental Biology in Cyanobacteria
MDPI AG, 2019Co-Authors: Antonia Herrero, Enrique FloresAbstract:Filamentous, Heterocyst-forming cyanobacteria are phototrophic multicellular organisms in which N2-fixing Heterocysts and CO2-fixing vegetative cells exchange regulators and nutrients [...
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overexpression of sepj alters septal morphology and Heterocyst pattern regulated by diffusible signals in anabaena
Molecular Microbiology, 2016Co-Authors: Vicente Mariscal, Antonia Herrero, Conrad W. Mullineaux, Dennis J Nurnberg, Enrique FloresAbstract: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.
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relationships between the abc exporter hetc and peptides that regulate the spatiotemporal pattern of Heterocyst distribution in anabaena
PLOS ONE, 2014Co-Authors: Laura Corralesguerrero, Enrique Flores, Antonia HerreroAbstract:In the model cyanobacterium Anabaena sp. PCC 7120, cells called Heterocysts that are specialized in the fixation of atmospheric nitrogen differentiate from vegetative cells of the filament in the absence of combined nitrogen. Heterocysts follow a specific distribution pattern along the filament, and a number of regulators have been identified that influence the Heterocyst pattern. PatS and HetN, expressed in the differentiating cells, inhibit the differentiation of neighboring cells. At least PatS appears to be processed and transferred from cell to cell. HetC is similar to ABC exporters and is required for differentiation. We present an epistasis analysis of these regulatory genes and of genes, hetP and asr2819, successively downstream from hetC, and we have studied the localization of HetC and HetP by use of GFP fusions. Inactivation of patS, but not of hetN, allowed differentiation to proceed in a hetC background, whereas inactivation of hetC in patS or patS hetN backgrounds decreased the frequency of contiguous proHeterocysts. A HetC-GFP protein is localized to the Heterocysts and especially near their cell poles, and a putative HetC peptidase domain was required for Heterocyst differentiation but not for HetC-GFP localization. hetP is also required for Heterocyst differentiation. A HetP-GFP protein localized mostly near the Heterocyst poles. ORF asr2819, which we denote patC, encodes an 84-residue peptide and is induced upon nitrogen step-down. Inactivation of patC led to a late spreading of the Heterocyst pattern. Whereas HetC and HetP appear to have linked functions that allow Heterocyst differentiation to progress, PatC may have a role in selecting sites of differentiation, suggesting that these closely positioned genes may be functionally related.
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compartmentalized cyanophycin metabolism in the diazotrophic filaments of a Heterocyst forming cyanobacterium
Proceedings of the National Academy of Sciences of the United States of America, 2014Co-Authors: Mireia Burnat, Antonia Herrero, Enrique FloresAbstract:Heterocyst-forming cyanobacteria are multicellular organisms in which growth requires the activity of two metabolically interdependent cell types, the vegetative cells that perform oxygenic photosynthesis and the dinitrogen-fixing Heterocysts. Vegetative cells provide the Heterocysts with reduced carbon, and Heterocysts provide the vegetative cells with fixed nitrogen. Heterocysts conspicuously accumulate polar granules made of cyanophycin [multi-L-arginyl-poly (L-aspartic acid)], which is synthesized by cyanophycin synthetase and degraded by the concerted action of cyanophycinase (that releases β-aspartyl-arginine) and isoaspartyl dipeptidase (that produces aspartate and arginine). Cyanophycin synthetase and cyanophycinase are present at high levels in the Heterocysts. Here we created a deletion mutant of gene all3922 encoding isoaspartyl dipeptidase in the model Heterocyst-forming cyanobacterium Anabaena sp. strain PCC 7120. The mutant accumulated cyanophycin and β-aspartyl-arginine, and was impaired specifically in diazotrophic growth. Analysis of an Anabaena strain bearing an All3922-GFP (green fluorescent protein) fusion and determination of the enzyme activity in specific cell types showed that isoaspartyl dipeptidase is present at significantly lower levels in Heterocysts than in vegetative cells. Consistently, isolated Heterocysts released substantial amounts of β-aspartyl-arginine. These observations imply that β-aspartyl-arginine produced from cyanophycin in the Heterocysts is transferred intercellularly to be hydrolyzed, producing aspartate and arginine in the vegetative cells. Our results showing compartmentalized metabolism of cyanophycin identify the nitrogen-rich molecule β-aspartyl-arginine as a nitrogen vehicle in the unique multicellular system represented by the Heterocyst-forming cyanobacteria.
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FraH Is Required for Reorganization of Intracellular Membranes during Heterocyst Differentiation in Anabaena sp. Strain PCC 7120
Journal of bacteriology, 2011Co-Authors: Victoria Merino-puerto, Antonia Herrero, Iris Maldener, Vicente Mariscal, Heinz Schwarz, Conrad W. Mullineaux, Enrique FloresAbstract:In the filamentous, Heterocyst-forming cyanobacteria, two different cell types, the CO(2)-fixing vegetative cells and the N(2)-fixing Heterocysts, exchange nutrients and regulators for diazotrophic growth. In the model organism Anabaena sp. strain PCC 7120, inactivation of fraH produces filament fragmentation under conditions of combined nitrogen deprivation, releasing numerous isolated Heterocysts. Transmission electron microscopy of samples prepared by either high-pressure cryo-fixation or chemical fixation showed that the Heterocysts of a ΔfraH mutant lack the intracellular membrane system structured close to the Heterocyst poles, known as the honeycomb, that is characteristic of wild-type Heterocysts. Using a green fluorescent protein translational fusion to the carboxyl terminus of FraH (FraH-C-GFP), confocal microscopy showed spots of fluorescence located at the periphery of the vegetative cells in filaments grown in the presence of nitrate. After incubation in the absence of combined nitrogen, localization of FraH-C-GFP changed substantially, and the GFP fluorescence was conspicuously located at the cell poles in the Heterocysts. Fluorescence microscopy and deconvolution of images showed that GFP fluorescence originated mainly from the region next to the cyanophycin plug present at the Heterocyst poles. Intercellular transfer of the fluorescent tracers calcein (622 Da) and 5-carboxyfluorescein (374 Da) was either not impaired or only partially impaired in the ΔfraH mutant, suggesting that FraH is not important for intercellular molecular exchange. Location of FraH close to the honeycomb membrane structure and lack of such structure in the ΔfraH mutant suggest a role of FraH in reorganization of intracellular membranes, which may involve generation of new membranes, during Heterocyst differentiation.
John C Meeks - One of the best experts on this subject based on the ideXlab platform.
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global transcription profiles of the nitrogen stress response resulting in Heterocyst or hormogonium development in nostoc punctiforme
Journal of Bacteriology, 2011Co-Authors: Harry D Christman, Elsie L Campbell, John C MeeksAbstract:The filamentous cyanobacterium Nostoc punctiforme differentiates from vegetative cells into three distinct cell types, Heterocysts, hormogonia, and akinetes, in response to different stimuli. Cultures growing with ammonium can be induced to form hormogonia or Heterocysts upon removal of the combined nitrogen. A DNA microarray consisting of 94% of the open reading frames predicted from the 9.059-Mb N. punctiforme genome was used to generate a global transcription data set consisting of seven time points over a 24-h period of nitrogen deprivation, which results in Heterocyst formation. This data set was compared to a similarly generated data set of nitrogen-starved N. punctiforme resulting in hormogonium formation that had previously been published (E. L. Campbell, H. Christman, and J. C. Meeks, J. Bacteriol. 190:7382-7391, 2008). The transition from vegetative cells to either Heterocysts or hormogonia resulted in rapid and sustained expression of genes required for utilization of alternate nitrogen sources. Overall, 1,036 and 1,762 genes were found to be differentially transcribed during the Heterocyst and hormogonium time courses, respectively, as analyzed with the Bayesian user-friendly software for analyzing time series microarray experiments (BATS). Successive transcription of Heterocyst regulatory, structural, and functional genes occurred over the 24 h required to form a functional Heterocyst. During hormogonium differentiation, some Heterocyst structural and functional genes were upregulated, while the Heterocyst master regulator hetR was downregulated. There are commonalities in differential expression between cells bound for differentiation into Heterocysts or hormogonia, yet the two paths are distinguished by their developmentally specific transcription profiles.
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the hetf gene product is essential to Heterocyst differentiation and affects hetr function in the cyanobacterium nostoc punctiforme
Journal of Bacteriology, 2001Co-Authors: Francis C Y Wong, John C MeeksAbstract:A novel gene, hetF, was identified as essential for Heterocyst development in the filamentous cyanobacterium Nostoc punctiforme strain ATCC 29133. In the absence of combined nitrogen, hetF mutants were unable to differentiate Heterocysts, whereas extra copies of hetF in trans induced the formation of clusters of Heterocysts. Sequences hybridizing to a hetF probe were detected only in Heterocyst-forming cyanobacteria. The inactivation and multicopy effects of hetF were similar to those of hetR, which encodes a self-degrading serine protease thought to be a central regulator of Heterocyst development. Increased transcription of hetR begins in developing cells 3 to 6 h after deprivation for combined nitrogen (N step-down), and the HetR protein specifically accumulates in Heterocysts. In the hetF mutant, this increase in hetR transcription was delayed, and a hetR promoter::green fluorescent protein (GFP) transcriptional reporter indicated that increased transcription of hetR occurred in all cells rather than only in developing Heterocysts. When a fully functional HetR-GFP fusion protein was expressed in the hetF mutant from a multicopy plasmid, HetR-GFP accumulated nonspecifically in all cells under nitrogen-replete conditions; when expressed in the wild type, HetR-GFP was observed only in Heterocysts after N step-down. HetF therefore appears to cooperate with HetR in a positive regulatory pathway and may be required for the increased transcription of hetR and localization of the HetR protein in differentiating Heterocysts.