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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.
Hebbar PB, Curtis SE (2000) Characterization of devH, a gene encoding a putative DNA binding protein required for heterocyst function in Anabaena sp. strain PCC 7120. J Bacteriol 182: 3572–3581. pmid:10852891 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. KUALA LUMPUR: Malaysia’s Hydrogen Economy and Technology Roadmap (HETR) has been launched, pushing the country to the forefront of energy transition and to becoming a regional leader in the renewable energy (RE) industry. To get further insights into HetL function, we wondered whether its activity would be mediated by its direct interaction with HetR. To test this, we used the bacterial two hybrid assay (BACTH), which is based on the reconstitution of adenylate cyclase (CyA) activity by two interacting proteins that bring the T18 and T25 domains of CyA into close proximity ( Karimova et al., 1998). T18 and T25 domains were fused to HetR and HetL proteins at their N-terminal extremities, and the dimerization ability of HetR was used as an internal control for this assay. The data in Figure 1B show that HetL displays a strong interaction with HetR. Interestingly, it seems that the HetR-hood domain is sufficient to mediate the interaction of HetR to HetL. Furthermore, this experiment indicated that HetL is able to form dimers (or oligomers) ( Figure 1B). 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.
Introduction
Ramaswamy KS, Carrasco CD, Fatma T, Golden JW: Cell-type specificity of the Anabaena fdxN-element rearrangement requires xisH and xisI. Mol Microbiol. 1997, 23 (6): 1241-1249. 10.1046/j.1365-2958.1997.3081671.x. The Creative Commons Public Domain Dedication waiver ( https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. We start by looking at the transcription of ntcA, which is regulated by HetR and NtcA. We assume that the probability that NtcA binds the promoter in the absence of 2-OG can be neglected. Taking into account that both HetR and NtcA dimerize to bind DNA we find:
Fig 5. States of a cyanobacterium when subjected to different conditions of nitrogen and diffusion. 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 Lopez-Igual R, Flores E, Herrero A: Inactivation of a heterocyst-specific invertase indicates a principal role of sucrose catabolism in heterocysts of Anabaena sp. J Bacteriol. 2010, 192 (20): 5526-5533. 10.1128/JB.00776-10. Young-Robbins SS, Risser DD, Moran JR, Haselkorn R, Callahan SM: Transcriptional regulation of the heterocyst patterning gene patA from Anabaena sp. strain PCC 7120. J Bacteriol. 2010, 192 (18): 4732-4740. 10.1128/JB.00577-10.Cells were then spun down at 4,000 × g for 5 minutes at 4°C and washed twice in 30 ml ice cold PBS (137 mM NaCl, 2 mM KCl, 10 mM Na 2HPO 4, 1.8 mM KH 2PO 4, pH 7.4). Washed and fixed pellets were resuspended in 500 μL ice-cold binding/wash buffer (100 mM NaHPO 4, 600 mM NaCl, 0.02% Tween 20, 1 EDTA Proteinase Inhibitor Tab, Roche Biosciences, in 10 ml total volume) on ice. Protein was extracted by bead beating 2 × 5 minutes with 2 minutes on ice in between. Complete lysis was confirmed by microscopy. Lysed cells were separated from beads via centrifugation and DNA was sheared via sonication on ice, 12 cycles of 20 seconds on, 15 second off at 14% power. Cell debris was pelleted via two cycles of centrifugation at 14,000 × g for 15 minutes at 4°C. Protein concentration was determined by absorbance at 280 nm and normalized to 20 mg/mL for each sample by dilution in cold binding/wash buffer. WT control cells were collected and processed in parallel with the HetR-6xHis cells except that the WT cells were collected at eight hours after nitrogen deprivation because they were being used to control for additional ChIP-seq samples collected at different times. Due to the fast dynamics that HetR and NtcA exhibit, we can approach the treatment of the system by adopting a point of view that follows the slower variables q s and q n. From this viewpoint, the time-evolution of the pair ( q s( t), q n( t)) is considered by assuming that q r and q a instantaneously relax to an equilibrium, which corresponds to a sink ( q r *, q a *) for the fixed pair ( q s( t), q n( t)). Depending on the region of the ( q s, q n)-plane, there are three fixed points (two sinks corresponding to the highest and the lowest concentrations respectively and a saddle in the middle) or one (a sink) for q r and q a (I and II). There are two one-sink regions that are separated from the two-sink region by saddle-node bifurcations (A-F). Sinks and saddles are represented by filled and unfilled circles respectively and arrows indicate the flow of the dynamics. We can then imagine the dynamics of q s and q n as evolving either in the bottom or in the top branch of I. In the two-sink region, both branches are plausible and the history of the dynamics determine the solution (hysteresis effect): a dynamics in a branch will continue in it until experiencing a bifurcation in the ( q r, q a) plane (see Fig. 5 for examples). Laurent S, Chen H, Bédu S, Ziarelli F, Peng L, Zhang CC (2005) Nonmetabolizable analogue of 2-oxoglutarate elicits heterocyst differentiation under repressive conditions in Anabaena sp. PCC 7120. P Natl Acad Sci USA 102: 9907–9912. Muro-Pastor MI, Reyes JC, Florencio FJ (2005) Ammonium assimilation in cyanobacteria. Photosynth Res 83: 135–150. pmid:16143848 Borthakur PB, Orozco CC, Young-Robbins SS, Haselkorn R, Callahan SM: Inactivation of patS and hetN causes lethal levels of heterocyst differentiation in the filamentous cyanobacterium Anabaena sp. PCC 7120. Mol Microbiol. 2005, 57 (1): 111-123. 10.1111/j.1365-2958.2005.04678.x.
This work did not involve human subjects, human material, or human data and therefore does not require ethical approval. Availability of supporting data Ehira S, Ohmori M (2006) NrrA directly regulates expression of hetR during heterocyst differentiation in the cyanobacterium Anabaena sp. strain PCC 7120. J Bacteriol 188: 8520–8525. pmid:17041048 Rectangular boxes represent genes ( ntcA, hetR and patS) while rounded boxes and circles represent transcription factors (NtcA, HetR and PatS) and smaller molecules (2-OG and cN) respectively. Normal-tipped and flat-tipped arrows stand for up-regulating and down-regulating processes respectively. Dashed lines stand for indirect or imperfectly understood interactions. The accumulation of 2-OG enhances the DNA-binding activity of NtcA, which in turn up-regulates the transcription of ntcA and hetR. HetR activates ntcA and hetR (composing the central NtcA-HetR autoregulatory loop), the inhibitor patS and other genes that lead to nitrogen fixation and the morphological changes involved in heterocyst differentiation. 2-OG and cN levels are linked through the GS/GOGAT cycle (see Fig. 2). Koch A, Meinhardt H (1994) Biological pattern formation: from basic mechanisms to complex structures. Rev Mod Phys 66: 1481–1511.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.
Heterocyst pattern was first speculated to be controlled by a pentapeptide RGSGR (designated as PatS5), a motif which was identified first as the C-terminal five residues of PatS 16 and later within a putative ketoacyl reductase HetN 17. Binding of PatS5 to HetR may abolish the DNA-binding capacity of HetR 11, a proposed mechanism to prevent the occurrence of multiple contiguous heterocysts ( Fig. 1). The in vitro binding assays suggested that two PatS5 molecules bind to a HetR homodimer at a dissociation constant K d of 227 nM 18, most likely at the proximity of the C-terminal region 18. Notably, HetR has a higher affinity towards a hexapeptide ERGSGR (termed PatS6, at a K d of 7 nM), compared to that of PatS5 19. However, the in vivo experiments suggested that the last eight residues could recreate the full activity of the native PatS 20. Thus the bona fide active form of PatS under physiological conditions remains unknown. By reducing the flow of cN from the exterior of the cell ( l n = 0) we find that a stable fixed point appears in the upper branch, a heterocyst state, while the vegetative state gets closer to the bifurcation region, thus becoming more susceptible to perturbations that can make the system reach the upper branch. In the absence of cN, the cyanobacterium would evolve from state A to state B in the lower branch until a perturbation pushes it to the upper branch, eventually becoming an heterocyst due to the field acting on that branch ( Fig. 5B and C).A landmark process of (prokaryotic) cellular differentiation and cooperative pattern formation is the heterocyst differentiation in cyanobacteria filaments [ 3, 4]. Cyanobacteria are one of the first organisms that developed multicellularity some (2–3) billion years ago [ 5]. These bacteria perform oxygenic photosynthesis releasing oxygen to the environment. However, nitrogenase, the enzyme that performs nitrogen fixation, is deactivated by oxygen so that nitrogen fixation cannot occur in its presence [ 6]. Cyanobacteria solve the incompatibility of incorporating both oxygenic photosynthesis and nitrogen fixation by separating these processes ( i) temporally, such as in the unicellular Cyanothece sp. strain ATCC 51142, which presents photosynthetic activity during the day and fixes nitrogen during the night [ 7], or ( ii) spatially, by the generation of non-photosynthetic nitrogen-fixing cells distributed along the filament and acting as nitrogen suppliers. 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 He said that IGEM 2023, which is held from Oct 4 to Oct 6, and themed “Race Towards Net Zero: Leadership for Climate Action”, could play a decisive leadership role in accelerating and delivering the region’s net zero and energy transition agenda. Alfonso M, Kirilovsky D (2001) Redox Control of ntcA Gene Expression in Synechocystis sp. PCC 6803. Nitrogen Availability and NtcA Protein 1. Plant Physiol 125: 969–981. pmid:11161053
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