Corral Higa, K. C. et al. The RGSGR amino acid motif of the intercellular signalling protein, HetN, is required for patterning of heterocysts in Anabaena sp. strain PCC 7120. Mol. Microbiol. 83, 682–93 (2012).

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.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. Cyanobacteria are the only bacteria on Earth able to draw their energy directly from the sun in the same way that plants do. In addition, some strains are able to ‘fix’ the nitrogen present in the atmosphere: they can extract this gas and transform it into nitrogen-based compounds necessary for life. However, both processes cannot happen in a given cell at the same time.

Valladares A, Muro-pastor AM, Herrero A, Flores E (2004) The NtcA-Dependent P 1 Promoter Is Utilized for glnA Expression in N 2—Fixing Heterocysts of Anabaena sp. Strain PCC 7120. J Bacteriol 186: 7337–7343. pmid:15489445

Discussion

We appreciate the concern. In the original Figure 7, we decided to present the diffusion of PatS from only one part of the differentiating cell which is incomplete and confusing for a reader non-familiar with the differentiation process. In the revised Figure 7, we corrected this point by drawing the diffusion of PatS from both sides of the heterocyst.

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. Meinhardt, H. Models of biological pattern formation: From elementary steps to the organization of embryonic axes. Curr. Top. Dev. Biol. 81, 1–63 (2008). 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. Corrales-Guerrero L, Mariscal V, Flores E, Herrero A: Functional dissection and evidence for intercellular transfer of the heterocyst-differentiation PatS morphogen. Mol Microbiol. 2013, 88 (6): 1093-1105. 10.1111/mmi.12244.

Author Contributions

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.

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.

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. With the implementation of initiatives under the recently unveiled National Energy Transition Roadmap (NETR) and (now) the HETR, Malaysia is set to be at the forefront of becoming a regional leader in the energy transition and renewable energy industry,” he said at the launch of IGEM 2023. Murray JD (2003) Mathematical Biology II: Spatial Models and Biomedical Applications computational model predicts phenotype from genotype. New York: Springer-Verlag, third edition. 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. X-ray diffraction data were collected at 100 K in a liquid nitrogen stream, using beamline BL17U with a Q315r CCD (ADSC, MARresearch, Germany) at the Shanghai Synchrotron Radiation Facility (SSRF). All diffraction data were integrated and scaled with the program HKL2000 35. Structure determination and refinement