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Taton, A, Unglaub F, Wright NE, Zeng WY, Paz-Yepes J, Brahamsha B, Palenik B, Peterson TC, Haerizadeh F, Golden SS, Golden JW.  2014.  Broad-host-range vector system for synthetic biology and biotechnology in cyanobacteria. Nucleic Acids Research. 42   10.1093/nar/gku673   AbstractWebsite

Inspired by the developments of synthetic biology and the need for improved genetic tools to exploit cyanobacteria for the production of renewable bio-products, we developed a versatile platform for the construction of broad-host-range vector systems. This platform includes the following features: (i) an efficient assembly strategy in which modules released from 3 to 4 donor plasmids or produced by polymerase chain reaction are assembled by isothermal assembly guided by short GC-rich overlap sequences. (ii) A growing library of molecular devices categorized in three major groups: (a) replication and chromosomal integration; (b) antibiotic resistance; (c) functional modules. These modules can be assembled in different combinations to construct a variety of autonomously replicating plasmids and suicide plasmids for gene knockout and knockin. (iii) A web service, the CYANO-VECTOR assembly portal, which was built to organize the various modules, facilitate the in silico construction of plasmids, and encourage the use of this system. This work also resulted in the construction of an improved broad-host-range replicon derived from RSF1010, which replicates in several phylogenetically distinct strains including a new experimental model strain Synechocystis sp. WHSyn, and the characterization of nine antibiotic cassettes, four reporter genes, four promoters, and a ribozyme-based insulator in several diverse cyanobacterial strains.

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Bradley, JM, Svistunenko DA, Pullin J, Hill N, Stuart RK, Palenik B, Wilson MT, Hemmings AM, Moore GR, Le Brun NE.  2019.  Reaction of O-2 with a diiron protein generates a mixed-valent Fe2+/Fe3+ center and peroxide. Proceedings of the National Academy of Sciences of the United States of America. 116:2058-2067.   10.1073/pnas.1809913116   AbstractWebsite

The gene encoding the cyanobacterial ferritin SynFtn is up-regulated in response to copper stress. Here, we show that, while SynFtn does not interact directly with copper, it is highly unusual in several ways. First, its catalytic diiron ferroxidase center is unlike those of all other characterized prokaryotic ferritins and instead resembles an animal H-chain ferritin center. Second, as demonstrated by kinetic, spectro-scopic, and high-resolution X-ray crystallographic data, reaction of O-2 with the di-Fe2+ center results in a direct, one-electron oxidation to a mixed-valent Fe2+/Fe3+ form. Iron-O-2 chemistry of this type is currently unknown among the growing family of proteins that bind a diiron site within a four alpha-helical bundle in general and ferritins in particular. The mixed-valent form, which slowly oxidized to the more usual di-Fe3+ form, is an intermediate that is continually generated during mineralization. Peroxide, rather than superoxide, is shown to be the product of O-2 reduction, implying that ferroxidase centers function in pairs via long-range electron transfer through the protein resulting in reduction of O-2 bound at only one of the centers. We show that electron transfer is mediated by the transient formation of a radical on Tyr40, which lies similar to 4 angstrom from the diiron center. As well as demonstrating an expansion of the iron-O-2 chemistry known to occur in nature, these data are also highly relevant to the question of whether all ferritins mineralize iron via a common mechanism, providing unequivocal proof that they do not.