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The inhibitor PatS is regulated by HetR, and we assume no other influence. For simplicity, we implicitly include the effect of HetN in PatS, as their action is expected to be equivalent (see the previous section). This gives the simple transcription velocity: 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. Wolk, C. P. Physiological basis of the pattern of vegetative growth of a blue-green alga. Proc. Natl. Acad. Sci. USA 57, 1246–51 (1967).

Meinhardt, H. Models of biological pattern formation: From elementary steps to the organization of embryonic axes. Curr. Top. Dev. Biol. 81, 1–63 (2008). SARS-CoV-2 spike protein plays a key role in mediating viral entry and inducing host immune responses. It can adopt either an open or closed conformation based on the position of its receptor-binding domain (RBD). It is yet unclear what cause these conformational changes or how they influence the spike's functions. Here we show that Lys417 in the RBD plays dual roles in the spike's structure: it stabilizes the closed conformation of the trimeric spike by mediating inter-spike-subunit interactions; it also directly interacts with ACE2 receptor. Hence, a K417V mutation has opposing effects on the spike's function: it opens up the spike for better ACE2 binding while weakening the RBD's direct binding to ACE2. The net outcomes of this mutation are to allow the spike to bind ACE2 with higher probability, mediate viral entry more efficiently, but become more exposed to neutralizing antibodies. Given that residue 417 has been a viral mutational hotspot, SARS-CoV-2 may have been evolving to strike a balance between infection potency and immune evasion, contributing to its pandemic spread.

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Wisén S, Jiang F, Bergman B, Mannervik B (1999) Expression and purification of the transcription factor NtcA from the cyanobacterium Anabaena PCC 7120. Protein expres purif 17: 351–357. Vijayan V, Jain IH, O’Shea EK: A high resolution map of a cyanobacterial transcriptome. Genome Biol. 2011, 12 (5): R47-10.1186/gb-2011-12-5-r47. Ramasubramanian TS, Wei TF, Golden JW (1994) Two Anabaena sp. strain PCC 7120 DNA-binding factors interact with vegetative cell- and heterocyst-specific genes. J Bacteriol 176: 1214–1223. pmid:8113160 Videau, P. et al. Expanding the Direct HetR Regulon in Anabaena sp. Strain PCC 7120. J. Bacteriol. 196, 1113–21 (2014).

The hetR gene from Anabaena sp. PCC 7120 was cloned into a modified pET28 vector with a six-histidine tag at the N-terminus. The DNA sequence encoding HetR Hood (Asp219-Asp299) was also cloned into this pET28a-derived vector. The two recombinant plasmids were validated by DNA sequencing (Sangon Biotech, Shanghai). HetR and HetR Hood proteins were overexpressed in Escherichia coli strain Rosetta (DE3) (Novagen, Madison). Cells were grown in 2 × YT medium (5 g NaCl, 16 g Bacto-Tryptone and 10 g yeast extract per liter) at 37 °C containing kanamycin and chloramphenicol at 30 and 34 μg/ml, respectively. At an OD 600 nm of 0.8, protein expression was induced with 0.2 mM isopropyl β-D-1-thiogalactopyranoside at 16 °C for 20 hr. Cells were harvested by centrifugation (6,000 × g, 4 °C, 10 min) and resuspended in 40 ml lysis buffer (1 M NaCl, 10 mM Tris-Cl, pH 7.8). After 5 min of sonication and centrifugation at 12,000 × g for 30 min, the supernatant containing the soluble target protein was pooled and loaded onto a Ni-NTA column (Qiagen, Mississauga, ON) equilibrated with the binding buffer (1 M NaCl, 10 mM Tris-Cl, pH 7.8). The target protein was eluted with 300 mM imidazole and further applied to a Superdex 75 column (GE Healthcare, UK) equilibrated with the binding buffer. The purity of protein was assessed by gel electrophoresis and the protein sample was stored at −80 °C. The work is structured as follows. First we present the main actors of the basic regulatory network and the different dynamical interactions that take place during the differentiation process. Then we develop a mathematical model for the unicellular reaction to nitrogen deprivation. Although a single cell model cannot provide a complete understanding of heterocyst formation, we analyze the main features that arise from the dynamical behavior of the system to gain insight about cell dynamics under different external conditions.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). Laurent, S. et al. Nonmetabolizable analogue of 2-oxoglutarate elicits heterocyst differentiation under repressive conditions in Anabaena sp. PCC 7120. Proc. Natl. Acad. Sci. USA 102, 9907–12 (2005). Here, Xu et al. show that HetL cannot diffuse from one cell to the other, and that it binds to HetR at the same place than PatS does. When both PatS and HetL are present, they compete to attach to HetR, which stops PatS from turning off HetR and deactivating the nitrogen-fixing program.

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. Zhou R, Wei X, Jiang N, Li H, Dong Y, Hsi KL, et al. (1998) Evidence that HetR protein is an unusual serine-type protease. P Natl Acad Sci USA 95: 4959–4963. A strain of cyanobacteria called Nostoc PCC 7120 can organise itself into long filaments of interconnected cells. Under certain conditions, one in every ten cells stops drawing its energy from the sun, and starts fixing atmospheric nitrogen instead. Exactly how the bacteria are able to ‘count to ten’ and organize themselves in such a precise pattern is still unclear. 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. Feldmann, E. A. et al. Differential binding between PatS C-terminal peptide fragments and HetR from Anabaena sp. PCC 7120. Biochemistry 51, 2436–42 (2012).

Correction

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 Flaherty BL, Van Nieuwerburgh F, Head SR, Golden JW: Directional RNA deep sequencing sheds new light on the transcriptional response of Anabaena sp. strain PCC 7120 to combined-nitrogen deprivation. BMC Genomics. 2011, 12 (1): 332-10.1186/1471-2164-12-332.

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 OG and cN indirectly interact through the GS/GOGAT cycle. Glutamine is transformed into glutamate by means of 2-OG through the 2-OG amidotransferase (GOGAT) while cN converts glutamate into glutamine through the glutamine synthetase (GS). The importance of the cycle in heterocyst differentiation is twofold. From one side, it constitutes the early one-cell sensor to nitrogen starvation: the absence of cN breaks the cycle down and 2-OG starts to accumulate, whose action leads to the cascade of processes that provoke the differentiation (see Fig. 1). Additionally, later during the differentiation, it processes the cN created by the heterocysts decreasing the levels of 2-OG. The latter is crucial for the formation of the heterocyst pattern (see Fig. 3). Yoon HS, Golden JW: Heterocyst pattern formation controlled by a diffusible peptide. Science. 1998, 282 (5390): 935-938. 10.1126/science.282.5390.935. HetR regulates most processes of the genetic circuit. It governs, among others, the transcription of ntcA, patS, hep, hgl and nif genes that lead to most of structural changes of the cell and to nitrogen fixation. Table 1. Parameters for Eq. (11) that reproduce heterocyst formation under noisy conditions and pattern formation when PatS and cN diffuse along a filament of cyanobacteria.Fig 1. Main components and interactions involved in the reaction to combined nitrogen deprivation in cyanobacteria. 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. 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. The protein for crystallization was concentrated to 12 mg/ml by ultrafiltration (Millipore Amicon). The single-stranded DNA was synthesized by Sangon Biotech. Complementary DNA strands were heated at 95 °C for 5 min and then annealed slowly to room temperature. Prior to crystallization, DNA duplex and the recombinant HetR were mixed in a 1.1:1.0 molar ratio, whereas HetR Hood was incubated with 8 mM PatS6 (ERGSGR) synthesized by GL Biochem (Shanghai). All crystals were grown using the hanging drop vapor diffusion method at 13 °C. The nanopipetting was performed using the Mosquito nanoliter liquid handling system (TTP LabTech). The native HetR–DNA crystals were obtained against the reservoir solution of 0.3 M calcium acetate and 0.1 M Bicine, pH 9.0, while the SeMet HetR–DNA crystals were grown against 0.2 M magnesium acetate for two days. The native PatS6–HetR Hood crystals were obtained from 25% PEG 4000, 0.1 M sodium citrate, pH 5.6 and 0.2 M ammonium acetate. All the crystals were transferred to the cryoprotectant (reservoir solution supplemented with 30% glycerol) and flash-cooled in liquid nitrogen before data collection. Data collection and processing Flaherty, B. L., Johnson, D. & Golden, J. W. Deep sequencing of HetR-bound DNA reveals novel HetR targets in Anabaena sp. strain PCC7120. BMC Microbiol. 14, 255 (2014).

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