scholarly journals Phenotypic Assessment Suggests Multiple Start Codons for HetN, an Inhibitor of Heterocyst Differentiation, in Anabaena sp. Strain PCC 7120

2018 ◽  
Vol 200 (16) ◽  
Author(s):  
Orion S. Rivers ◽  
Silvia Beurmann ◽  
Allexa Dow ◽  
Loralyn M. Cozy ◽  
Patrick Videau
Keyword(s):  
De Novo ◽  
Mutational Analysis ◽  
Protein Localization ◽  
Periodic Pattern ◽  
Regulatory Sequences ◽  
Filamentous Cyanobacterium ◽  
Nitrogen Fixing ◽  
Content Type ◽  
Inhibitory Signal ◽  
Pcc 7120

ABSTRACT Multicellular organisms must carefully regulate the timing, number, and location of specialized cellular development. In the filamentous cyanobacterium Anabaena sp. strain PCC 7120, nitrogen-fixing heterocysts are interspersed between vegetative cells in a periodic pattern to achieve an optimal exchange of bioavailable nitrogen and reduced carbon. The spacing between heterocysts is regulated by the activity of two developmental inhibitors, PatS and HetN. PatS functions to create a de novo pattern from a homogenous field of undifferentiated cells, while HetN maintains the pattern throughout subsequent growth. Both PatS and HetN harbor the peptide motif ERGSGR, which is sufficient to inhibit development. While the small size of PatS makes the interpretation of inhibitory domains relatively simple, HetN is a 287-amino-acid protein with multiple functional regions. Previous work suggested the possibility of a truncated form of HetN containing the ERGSGR motif as the source of the HetN-derived inhibitory signal. In this work, we present evidence that the glutamate of the ERGSGR motif is required for proper HetN inhibition of heterocysts. Mutational analysis and subcellular localization indicate that the gene encoding HetN uses two methionine start codons (M1 and M119) to encode two protein forms: M1 is required for protein localization, while M119 is primarily responsible for inhibitory function. Finally, we demonstrate that patS and hetN are not functionally equivalent when expressed from the other gene's regulatory sequences. Taken together, these results help clarify the functional forms of HetN and will help refine future work defining a HetN-derived inhibitory signal in this model of one-dimensional periodic patterning. IMPORTANCE The proper placement of different cell types during a developmental program requires the creation and maintenance of a biological pattern to define the cells that will differentiate. Here we show that the HetN inhibitor, responsible for pattern maintenance of specialized nitrogen-fixing heterocyst cells in the filamentous cyanobacterium Anabaena, may be produced from two different start methionine codons. This work demonstrates that the two start sites are individually involved in a different HetN function, either membrane localization or inhibition of cellular differentiation.

scholarly journals Mutation of themurCandmurBGenes Impairs Heterocyst Differentiation in Anabaena sp. Strain PCC 7120

2016 ◽  
Vol 198 (8) ◽  
pp. 1196-1206 ◽  
Author(s):  
Patrick Videau ◽  
Orion S. Rivers ◽  
Blake Ushijima ◽  
Reid T. Oshiro ◽  
Min Joo Kim ◽  
...  
Keyword(s):  
Marker Gene ◽  
Periodic Pattern ◽  
Filamentous Cyanobacterium ◽  
Aberrant Expression ◽  
Content Type ◽  
Peptidoglycan Synthesis ◽  
Enzymatic Function ◽  
Cellular Integrity ◽  
Pcc 7120 ◽  
Heterocyst Development

ABSTRACTTo stabilize cellular integrity in the face of environmental perturbations, most bacteria, including cyanobacteria, synthesize and maintain a strong, flexible, three-dimensional peptidoglycan lattice.Anabaenasp. strain PCC 7120 is a filamentous cyanobacterium capable of differentiating morphologically distinct nitrogen-fixing heterocyst cells in a periodic pattern. While heterocyst development has been shown to require proper peptidoglycan remodeling, the role of peptidoglycan synthesis has remained unclear. Here we report the identification of two peptidoglycan synthesis genes,murC(alr5065) andmurB(alr5066), as required for heterocyst development. ThemurCandmurBgenes are predicted to encode a UDP-N-acetylmuramate:l-alanine ligase and a UDP-N-acetylenolpyruvoylglucosamine reductase, respectively, and we confirm enzymatic function through complementation ofEscherichia colistrains deficient for these enzymes. Cells depleted of eithermurCormurBexpression failed to differentiate heterocysts under normally inducing conditions and displayed decreased filament integrity. To identify the stage(s) of development affected bymurCormurBdepletion, the spatial distribution of expression of the patterning marker gene,patS, was examined. WhereasmurBdepletion did not affect the pattern ofpatSexpression,murCdepletion led to aberrant expression ofpatSin all cells of the filament. Finally, expression ofgfpcontrolled by the region of DNA immediately upstream ofmurCwas enriched in differentiating cells and was repressed by the transcription factor NtcA. Collectively, the data in this work provide evidence for a direct link between peptidoglycan synthesis and the maintenance of a biological pattern in a multicellular organism.IMPORTANCEMulticellular organisms that differentiate specialized cells must regulate morphological changes such that both cellular integrity and the dissemination of developmental signals are preserved. Here we show that the multicellular bacteriumAnabaena, which differentiates a periodic pattern of specialized heterocyst cells, requires peptidoglycan synthesis by the murine ligase genesmurC(alr5065) andmurB(alr5066) for maintenance of patterned gene expression, filament integrity, and overall development. This work highlights the significant influence that intracellular structure and intercellular connections can have on the execution of a developmental program.

scholarly journals Anabaena sp. Strain PCC 7120 hetY Gene Influences Heterocyst Development

2003 ◽  
Vol 185 (23) ◽  
pp. 6995-7000 ◽  
Author(s):  
Ho-Sung Yoon ◽  
Martin H. Lee ◽  
Jin Xiong ◽  
James W. Golden
Keyword(s):  
Atp Binding ◽  
Binding Motif ◽  
Filamentous Cyanobacterium ◽  
Hypothetical Proteins ◽  
Nitrogen Fixing ◽  
Fixed Nitrogen ◽  
Anabaena Sp ◽  
Time Required ◽  
Pcc 7120 ◽  
Heterocyst Development

ABSTRACT The filamentous cyanobacterium Anabaena (Nostoc) sp. strain PCC 7120 responds to starvation for fixed nitrogen by producing a semiregular pattern of nitrogen-fixing cells called heterocysts. Overexpression of the hetY gene partially suppressed heterocyst formation, resulting in an abnormal heterocyst pattern. Inactivation of hetY increased the time required for heterocyst maturation and caused defects in heterocyst morphology. The 489-bp hetY gene (alr2300), which is adjacent to patS (asl2301), encodes a protein that belongs to a conserved family of bacterial hypothetical proteins that contain an ATP-binding motif.

scholarly journals Sucrose Synthesis in the Nitrogen-Fixing Cyanobacterium Anabaena sp. Strain PCC 7120 Is Controlled by the Two-Component Response Regulator OrrA

2014 ◽  
Vol 80 (18) ◽  
pp. 5672-5679 ◽  
Author(s):  
Shigeki Ehira ◽  
Satoshi Kimura ◽  
Shogo Miyazaki ◽  
Masayuki Ohmori
Keyword(s):  
Salt Stress ◽  
Response Regulator ◽  
Sucrose Phosphate Synthase ◽  
Early Response ◽  
Sucrose Synthesis ◽  
Nitrogen Fixing ◽  
Content Type ◽  
Pcc 7120 ◽  
Sucrose Phosphate ◽  
Phosphate Synthase

ABSTRACTThe filamentous, nitrogen-fixing cyanobacteriumAnabaenasp. strain PCC 7120 accumulates sucrose as a compatible solute against salt stress. Sucrose-phosphate synthase activity, which is responsible for the sucrose synthesis, is increased by salt stress, but the mechanism underlying the regulation of sucrose synthesis remains unknown. In the present study, a response regulator, OrrA, was shown to control sucrose synthesis. Expression ofspsA, which encodes a sucrose-phosphate synthase, andsusAandsusB, which encode sucrose synthases, was induced by salt stress. In theorrAdisruptant, salt induction of these genes was completely abolished. The cellular sucrose level of theorrAdisruptant was reduced to 40% of that in the wild type under salt stress conditions. Moreover, overexpression oforrAresulted in enhanced expression ofspsA,susA, andsusB, followed by accumulation of sucrose, without the addition of NaCl. We also found that SigB2, a group 2 sigma factor of RNA polymerase, regulated the early response to salt stress under the control of OrrA. It is concluded that OrrA controls sucrose synthesis in collaboration with SigB2.

scholarly journals Sinorhizobium melilotiGlutathione Reductase Is Required for both Redox Homeostasis and Symbiosis

2017 ◽  
Vol 84 (3) ◽  
Author(s):  
Guirong Tang ◽  
Ningning Li ◽  
Yumin Liu ◽  
Liangliang Yu ◽  
Junhui Yan ◽  
...  
Keyword(s):  
Nitrogen Fixation ◽  
Sinorhizobium Meliloti ◽  
De Novo ◽  
Redox Homeostasis ◽  
Nodule Development ◽  
Glutathione Synthetase ◽  
Insertion Mutation ◽  
Nitrogen Fixing ◽  
Content Type ◽  
In Cells

ABSTRACTGlutathione (l-γ-glutamyl-l-cysteinylglycine) (GSH), one of the key antioxidants inSinorhizobium meliloti, is required for the development of alfalfa (Medicago sativa) nitrogen-fixing nodules. Glutathione exists as either reduced glutathione (GSH) or oxidized glutathione (GSSG), and its content is regulated by two pathways inS. meliloti. The first pathway is thede novosynthesis of glutathione from its constituent amino acids, namely, Glu, Cys, and Gly, catalyzed by γ-glutamylcysteine synthetase (GshA) and glutathione synthetase (GshB). The second pathway is the recycling of GSSG via glutathione reductase (GR). However, whether theS. melilotiGR functions similarly to GshA and GshB1 during symbiotic interactions with alfalfa remains unknown. In this study, a plasmid insertion mutation of theS. melilotigorgene, which encodes GR, was constructed, and the mutant exhibited delayed alfalfa nodulation, with 75% reduction in nitrogen-fixing capacity. Thegormutant demonstrated increased accumulation of GSSG and a decreased GSH/GSSG ratio in cells. The mutant also showed defective growth in rich broth and minimal broth and was more sensitive to the oxidants H2O2and sodium nitroprusside. Interestingly, the expression ofgshA,gshB1,katA, andkatBwas induced in the mutant. These findings reveal that the recycling of glutathione is important forS. melilotito maintain redox homeostasis and to interact symbiotically with alfalfa.IMPORTANCEThe antioxidant glutathione is regulated by its synthetase and reductase in cells. In the symbiotic bacteriumS. meliloti, thede novosynthesis of glutathione is essential for alfalfa nodulation and nitrogen fixation. In this study, we observed that the recycling of glutathione from GSSG not only was required for redox homeostasis and oxidative stress protection inS. meliloticells but also contributed to alfalfa nodule development and competition capacity. Our findings demonstrate that the recycling of glutathione plays a key role in nitrogen fixation symbiosis.

scholarly journals Inactivation of Three RG(S/T)GR Pentapeptide-Containing Negative Regulators of HetR Results in Lethal Differentiation of Anabaena PCC 7120

Life ◽  
2020 ◽  
Vol 10 (12) ◽  
pp. 326
Author(s):  
Ivan Khudyakov ◽  
Grigory Gladkov ◽  
Jeff Elhai
Keyword(s):  
Mutational Analysis ◽  
Filamentous Cyanobacterium ◽  
Intercellular Signaling ◽  
Tight Control ◽  
Negative Regulators ◽  
Short Peptide ◽  
Anabaena Pcc 7120 ◽  
Cyanobacterium Anabaena ◽  
Anabaena Sp ◽  
Pcc 7120

The filamentous cyanobacterium Anabaena sp. PCC 7120 produces, during the differentiation of heterocysts, a short peptide PatS and a protein HetN, both containing an RGSGR pentapeptide essential for activity. Both act on the master regulator HetR to guide heterocyst pattern formation by controlling the binding of HetR to DNA and its turnover. A third small protein, PatX, with an RG(S/T)GR motif is present in all HetR-containing cyanobacteria. In a nitrogen-depleted medium, inactivation of patX does not produce a discernible change in phenotype, but its overexpression blocks heterocyst formation. Mutational analysis revealed that PatX is not required for normal intercellular signaling, but it nonetheless is required when PatS is absent to prevent rapid ectopic differentiation. Deprivation of all three negative regulators—PatS, PatX, and HetN—resulted in synchronous differentiation. However, in a nitrogen-containing medium, such deprivation leads to extensive fragmentation, cell lysis, and aberrant differentiation, while either PatX or PatS as the sole HetR regulator can establish and maintain a semiregular heterocyst pattern. These results suggest that tight control over HetR by PatS and PatX is needed to sustain vegetative growth and regulated development. The mutational analysis has been interpreted in light of the opposing roles of negative regulators of HetR and the positive regulator HetL.

scholarly journals Temporal and Spatial Regulation of the Four Transcription Start Sites of hetR from Anabaena sp. Strain PCC 7120

2009 ◽  
Vol 192 (4) ◽  
pp. 1088-1096 ◽  
Author(s):  
Ramya Rajagopalan ◽  
Sean M. Callahan
Keyword(s):  
Pattern Formation ◽  
Fluorescent Protein ◽  
Single Cells ◽  
Genetic Network ◽  
Periodic Pattern ◽  
Regulation Of Transcription ◽  
Filamentous Cyanobacterium ◽  
Gfp Reporter ◽  
Translational Start ◽  
Pcc 7120

ABSTRACT The filamentous cyanobacterium Anabaena sp. strain PCC 7120 forms nitrogen-fixing heterocysts in a periodic pattern in response to combined-nitrogen limitation in the environment. The master regulator of heterocyst differentiation, HetR, is necessary for both pattern formation and commitment of approximately every 10th cell of a filament to differentiation into a heterocyst. In this study, the individual contributions of four transcriptional start points (tsps) in regulation of transcription of hetR were assessed, and the effects of the regulatory genes patS, hetN, and patA on transcription from the tsps were determined. The tsp located at nucleotide −271 relative to the translational start site (−271 tsp) was the most tightly regulated tsp, and fluorescence from a −271 tsp-green fluorescent protein (GFP) reporter fusion was observed initially in groups of two cells and later in single cells arranged in a spatial pattern that mimicked the pattern of heterocysts that emerged. Conversely, the fluorescence from the −184 and −728/−696 tsp-GFP reporter fusions was uniform throughout filaments. Transcription from the −271 tsp was severely downregulated in a strain in which the patA gene, which encodes a positive regulator of differentiation, was deleted, and it was not detectable in strains overexpressing the genes encoding the negative regulators of differentiation, patS and hetN. In strains lacking the −271 tsp of hetR, pattern formation, the timing of commitment to differentiation, and the number of cells that differentiated into heterocysts were affected. Taken together, these results demonstrate the role of regulation of the −271 tsp of hetR in the genetic network that governs heterocyst pattern formation and differentiation.

scholarly journals Transcriptional Regulation of the Heterocyst Patterning Gene patA from Anabaena sp. Strain PCC 7120

2010 ◽  
Vol 192 (18) ◽  
pp. 4732-4740 ◽  
Author(s):  
Shirley S. Young-Robbins ◽  
Douglas D. Risser ◽  
Jennifer R. Moran ◽  
Robert Haselkorn ◽  
Sean M. Callahan
Keyword(s):  
Cell Division ◽  
Periodic Pattern ◽  
Filamentous Cyanobacterium ◽  
Nitrogen Deprivation ◽  
Translational Start Site ◽  
Terminal Domain ◽  
Anabaena Sp ◽  
Altered Cell ◽  
Translational Start ◽  
Pcc 7120

ABSTRACT The filamentous cyanobacterium Anabaena sp. strain PCC 7120 forms a periodic pattern of nitrogen-fixing heterocysts when grown in the absence of combined nitrogen. PatA is necessary for proper patterning of heterocysts along filaments. In this study, apparent transcriptional start points (tsps) were identified at nucleotides −305, −614, and −645 relative to the translational start site (−305, −614, and −645 tsps). Transcriptional reporter fusions were used to show that transcription from the −305 tsp was induced in all cells of filaments in response to nitrogen deprivation, required hetR for induction, and increased in a patA mutant. Transcription from −614/−645 tsp reporter fusions was spatially regulated and occurred primarily in cells that would become heterocysts. Complementation of a patA mutant strain by alleles encoding substitutions in, or deletion of, the putative phosphoacceptor C-terminal domain indicates that the PATAN domain can function independently of the C-terminal domain of PatA. Localization of a ring of PatA-GFP at sites of cell division, as well as the formation of enlarged cells with altered cell morphology when patA was overexpressed, suggests that PatA may participate in cell division.

scholarly journals ABC Transporter Required for Intercellular Transfer of Developmental Signals in a Heterocystous Cyanobacterium

2015 ◽  
Vol 197 (16) ◽  
pp. 2685-2693 ◽  
Author(s):  
Patrick Videau ◽  
Orion S. Rivers ◽  
Kelly C. Higa ◽  
Sean M. Callahan
Keyword(s):  
Abc Transporter ◽  
Morphological Differentiation ◽  
Cellular Membrane ◽  
Periodic Pattern ◽  
Filamentous Cyanobacterium ◽  
Vegetative Cells ◽  
Content Type ◽  
Intercellular Transfer ◽  
Dependent Signal ◽  
And Function

ABSTRACTIn the filamentous cyanobacteriumAnabaena,patSandhetNencode peptide-derived signals with many of the properties of morphogens. These signals regulate the formation of a periodic pattern of heterocysts by lateral inhibition of differentiation. Here we show that intercellular transfer of thepatS- andhetN-dependent developmental signals from heterocysts to vegetative cells requires HetC, a predicted ATP-binding cassette transporter (ABC transporter). Relative to the wild type, in ahetCmutant differentiation resulted in a reduced number of heterocysts that were incapable of nitrogen fixation, but deletion ofpatSorhetNrestored heterocyst number and function in ahetCbackground. These epistasis results suggest that HetC is necessary for conferring self-immunity to the inhibitors on differentiating cells. Nine hours after induction of differentiation, HetC was required for neither induction of transcription ofpatSnor intercellular transfer of thepatS-encoded signal to neighboring cells. Conversely, in strains lacking HetC, thepatS- andhetN-encoded signals were not transferred from heterocyst cells to adjacent vegetative cells. The results support a model in which thepatS-dependent signal is initially transferred between vegetative cells in a HetC-independent fashion, but some time before morphological differentiation of heterocysts is complete, transfer of both signals transitions to a HetC-dependent process.IMPORTANCEHow chemical cues that regulate pattern formation in multicellular organisms move from one cell to another is a central question in developmental biology. In this study, we show that an ABC transporter, HetC, is necessary for transport of two developmental signals between different types of cells in a filamentous cyanobacterium. ABC transporters are found in organisms as diverse as bacteria and humans and, as the name implies, are often involved in the transport of molecules across a cellular membrane. The activity of HetC was shown to be required for signaling between heterocysts, which supply fixed nitrogen to the organism, and other cells, as well as for conferring immunity to self-signaling on developing heterocysts.

scholarly journals Transcriptional dynamics of developmental genes assessed with an FMN-dependent fluorophore in mature heterocysts of Anabaena sp. strain PCC 7120

Microbiology ◽  
2014 ◽  
Vol 160 (9) ◽  
pp. 1874-1881 ◽  
Author(s):  
Patrick Videau ◽  
Reid T. Oshiro ◽  
Loralyn M. Cozy ◽  
Sean M. Callahan
Keyword(s):  
De Novo ◽  
Flavin Mononucleotide ◽  
Filamentous Cyanobacterium ◽  
Reporter Expression ◽  
Transcriptional Fusion ◽  
Developmental Genes ◽  
Transcriptional Dynamics ◽  
Gfp Fluorescence ◽  
Anabaena Sp ◽  
Pcc 7120

Anabaena sp. strain PCC 7120 is a filamentous cyanobacterium that differentiates nitrogen-fixing heterocysts when available combined nitrogen is limiting. Growth under diazotrophic conditions results in a mixture of ‘new’ (recently differentiated) and ‘old’ (mature) heterocysts. The microoxic environment present in heterocysts makes the interpretation of gene expression using oxygen-dependent fluorophores, including GFP, difficult. The work presented here evaluates the transcriptional dynamics of three developmental genes in mature heterocysts utilizing EcFbFP, a flavin mononucleotide-dependent fluorophore, as the reporter. Expression of both GFP and EcFbFP from the heterologous petE promoter showed that, although GFP and EcFbFP fluoresced in both vegetative cells and new heterocysts, only EcFbFP fluoresced in old heterocysts. A transcriptional fusion of EcFbFP to the late-stage heterocyst-specific nifB promoter displayed continued expression beyond the cessation of GFP fluorescence in heterocysts. Promoter fusions of the master regulator of differentiation, hetR, and its inhibitors, patS and hetN, to GFP and EcFbFP were visualized to determine their role(s) in heterocyst function after morphogenesis. The expression of hetR and hetN was found to persist beyond the completion of development in most heterocysts, whereas patS expression ceased. These data are consistent with a model of heterocyst patterning in which patS is involved in de novo pattern formation, hetN is required for pattern maintenance, and hetR is needed for all stages of development.

scholarly journals De novo design of a reversible phosphorylation-dependent switch for membrane targeting

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Leon Harrington ◽  
Jordan M. Fletcher ◽  
Tamara Heermann ◽  
Derek N. Woolfson ◽  
Petra Schwille
Keyword(s):  
Protein Interactions ◽  
Lipid Membrane ◽  
De Novo ◽  
Protein Localization ◽  
Protein Structures ◽  
Spatiotemporal Pattern ◽  
Membrane Targeting ◽  
Protein Protein Interactions ◽  
Reversible Phosphorylation ◽  
Potential Applications

AbstractModules that switch protein-protein interactions on and off are essential to develop synthetic biology; for example, to construct orthogonal signaling pathways, to control artificial protein structures dynamically, and for protein localization in cells or protocells. In nature, the E. coli MinCDE system couples nucleotide-dependent switching of MinD dimerization to membrane targeting to trigger spatiotemporal pattern formation. Here we present a de novo peptide-based molecular switch that toggles reversibly between monomer and dimer in response to phosphorylation and dephosphorylation. In combination with other modules, we construct fusion proteins that couple switching to lipid-membrane targeting by: (i) tethering a ‘cargo’ molecule reversibly to a permanent membrane ‘anchor’; and (ii) creating a ‘membrane-avidity switch’ that mimics the MinD system but operates by reversible phosphorylation. These minimal, de novo molecular switches have potential applications for introducing dynamic processes into designed and engineered proteins to augment functions in living cells and add functionality to protocells.

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