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Gedtzen Z, Salgado J, Osses A, Asenjo J, Rapaport I, Andrews B (2009) Modeling heterocyst pattern formation in cyanobacteria. BMC Bioinf 10: S16. Purified HetR and mutants were dialyzed against a buffer containing 1 M NaCl, 5% (v/v) glycerol and 10 mM Tris-Cl, pH 7.8 for 12 hr. The data were collected on an iTC200 (MicroCal) at 25 °C by injecting an initial 0.4 μl aliquot and the following 19 consecutive 2 μl aliquots. The sample cell was loaded with 200 μl protein while the injection syringe was loaded with 40 μl PatS6. The wild-type HetR, mutants E254A and D256A were diluted to a final concentration of 10 μM, whereas the concentration of mutants R223W, E253A, D270A, D278A, D270A/D278A and HetR Hood was 50 μM. The concentration of PatS6 was 15 times (150 or 750 μM) to that of the full-length protein or 40 times (2 mM) to that of HetR Hood. EMSA assays To confirm this interaction, we developed a BioLayer interferometry (BLi) assay. For this purpose, HetR and HetL proteins were produced and purified using affinity chromatography ( Figure 1—figure supplement 1). HetR was biotinylated and immobilized on streptavidin biosensors as the ligand, while HetL was used as the analyte. Upon addition of HetL, a concentration-dependent association was recorded and decreased during the dissociation step corresponding to the washing of the sensor, indicating a direct interaction between HetL and HetR ( Figure 1C). The estimated dissociation constant (K D) of the HetR-HetL interaction was 6 µM. The interaction between HetR and HetL observed in the BACTH assay was thus confirmed by BLi. Khudyakov IY, Golden JW: Different functions of HetR, a master regulator of heterocyst differentiation in Anabaena sp. PCC 7120, can be separated by mutation. Proc Natl Acad Sci U S A. 2004, 101 (45): 16040-16045. 10.1073/pnas.0405572101.
NtcA presents autoregulation [ 22, 24, 25] and indirectly activates the key gene that controls cell differentiation and pattern formation: hetR [ 26– 28]. To bind DNA, NtcA needs to homodimerize [ 29, 30]. In conclusion, the accumulation of 2-OG is the factor that triggers differentiation. In agreement with this idea, artificial increased levels of 2-OG result in heterocyst development even in the presence of ammonium [ 16, 18, 31].
which constitutes the model for a cyanobacteria filament. To account for environment variability we add white noise, G i, *( t), of the same amplitude, ⟨ G i, *( t) G i, *( t ′)⟩ = ξδ( t − t ′), for all the components of the system. Based on these equations, we investigate the conditions that lead to a heterocyst pattern. It is easy to notice that they correspond to an activator-inhibitor system of cells coupled in a reaction-diffusion scheme [ 50]. This kind of system produces regular pattern formation [ 51– 53]. Turing (linear stability) analysis of equations (16) (see S2 text) provides insight on the periodicity of patterns. It is interesting to show that the minimum periodicity observed in such analysis is larger than 1, which means that a single bacteria is unable to differentiate. Distinct from the previously reported HTH motif 25, the DNA-binding motif of HetR is composed of three helices: the two canonical helices α4 and α5, in addition to an auxiliary helix α10 from the symmetric subunit. Moreover, both the flap and the hood domains adopt novel folds; thus HetR represents a novel transcription factor. The DNA-binding pattern of Anabaena HetR Aldea MR, Mella-Herrera RA, Golden JW: Sigma factor genes sigC, sigE, and sigG are upregulated in heterocysts of the cyanobacterium Anabaena sp. strain PCC 7120. J Bacteriol. 2007, 189 (22): 8392-8396. 10.1128/JB.00821-07.
Fiedler G, Muro-Pastor A (2001) NtcA-Dependent Expression of the devBCAOperon, Encoding a Heterocyst-Specific ATP-Binding Cassette Transporter in Anabaena spp. J Bacteriol 183: 3795–3799. pmid:11371545 The last stages of heterocyst development cause the physiological changes of the cell aimed at creating an anaerobic environment that sustains nitrogen fixation. To this end, two new membrane layers are biosynthesized to decrease the entry of oxygen into the cell [ 44]. The morphogenesis of these two layers is controlled by two family of genes, hep and hgl, that are indirectly up-regulated by HetR [ 35]. After these morphological changes the genes in charge of nitrogen fixation, nif genes, are expressed. These genes encode, among others, the enzyme nitrogenase, which ultimately performs nitrogen fixation.DNA was prepared for sequencing with the Illumina ChIP-seq Sample prep kit by the Next Generation Sequencing Core at The Scripps Research Institute (La Jolla, CA) following the manufacturer's protocol. Sequencing was performed on the Illumina HiSeq platform with 4 samples multiplexed on one cell, yielding approximately 40 million 40-bp reads per sample. Sequence reads were demultiplexed based on index sequences and saved as FASTA files for analysis in CLC Genomics Workbench 5. Sequence alignment and peak finding Buikema WJ, Haselkorn R: Characterization of a gene controlling heterocyst differentiation in the cyanobacterium Anabaena 7120. Genes Dev. 1991, 5 (2): 321-330. 10.1101/gad.5.2.321. Shi Y, Zhao W, Zhang W, Ye Z, Zhao J (2006) Regulation of intracellular free calcium concentration during heterocyst differenciation by HetR and NtcA in Anabaena sp. PCC7120. Proc Nat Acad Sci (USA) 103: 11334–11339. Greenside HS and Helfand E (1981) Numerical-Integration of Stochastic Differential Equations 2. Bell Syst Tech J 60: 1927–1940.
Higa KC, Callahan SM: Ectopic expression of hetP can partially bypass the need for hetR in heterocyst differentiation by Anabaena sp. strain PCC 7120. Mol Microbiol. 2010, 77 (3): 562-574. 10.1111/j.1365-2958.2010.07257.x. His-tagged HetR was bound to Dynabeads (Dynabeads His-tag Isolation and Pull-down beads, Invitrogen) following the manufacturer's protocol at 4°C and eluted in 100 μL elution buffer (100 mM imidazole, 50 mM NaPO 4, 300 mM NaCl, 0.01% Tween 20). Crosslinks were reversed at 65°C for 18 hours. The bound DNA size distribution was determined on a 1% agarose gel. IP efficiency was measured via western blotting of HetR-6xHis with the Qiagen Penta-His antibody, BSA Free. After crosslinks were reversed, proteins were digested by the addition of 250 μL TE, 4 μL of 20 μg/μL glycogen, and 10 μL of 10 μg/μL proteinase K for 2 hours at 37°C. DNA was column purified with the Promega SV DNA purification kit and resuspended in 30 μL nuclease free water. DNA library preparation and sequencing Purinergic signaling activated by extracellular nucleotides and their derivative nucleosides trigger sophisticated signaling networks. The outcome of these pathways determine the capacity of the organism to survive under challenging conditions. Both extracellular ATP (eATP) and Adenosine (eAdo) act as primary messengers in mammals, essential for immunosuppressive responses. Despite the clear role of eATP as a plant damage-associated molecular pattern, the function of its nucleoside, eAdo, and of the eAdo/eATP balance in plant stress response remain to be fully elucidated. This is particularly relevant in the context of plant-microbe interaction, where the intruder manipulates the extracellular matrix. Here, we identify Ado as a main molecule secreted by the vascular fungus Fusarium oxysporum. We show that eAdo modulates the plant's susceptibility to fungal colonization by altering the eATP-mediated apoplastic pH homeostasis, an essential physiological player during the infection of this pathogen. Our work indicates that plant pathogens actively imbalance the apoplastic eAdo/eATP levels as a virulence mechanism.Mitchison GJ, Smith RJ, Road H (1973) Pattern formation in the blue-green alga, anabaena. I. Basic Mechanisms. J Cell Sci 12: 707–723. pmid:4198321
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