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  • Title: Development of replicative oriC plasmids and their versatile use in genetic manipulation of Cytophaga hutchinsonii.
    Author: Xu Y, Ji X, Chen N, Li P, Liu W, Lu X.
    Journal: Appl Microbiol Biotechnol; 2012 Jan; 93(2):697-705. PubMed ID: 21935590.
    Abstract:
    Cytophaga hutchinsonii is a Gram-negative aerobic soil bacterium which can digest crystalline cellulose completely through a strategy different from that of the well-studied cellulolytic aerobic fungi and anaerobic bacteria. However, despite the availability of a published genome sequence, studies on this organism have been very limited because of the lack of a genetic manipulation system. This paper describes the development of replicative oriC plasmids, carrying the replication origin of the C. hutchinsonii chromosome, and an electroporation method for Escherichia coli-C. hutchinsonii shuttle vectors based on oriC plasmids with an efficiency of about 2 × 10(4) transformants per microgram plasmid DNA. Heterologous proteins, including green fluorescent protein and β-galactosidase, were expressed successfully and proved functional in C. hutchinsonii under the control of the CHU_1284 promoter in oriC plasmids. Finally, the gene CHU_0344, encoding the main extracellular protein, was targeted by homologous recombination based on the oriC plasmid. These genetic techniques and tools will provide a means to study the novel cellulose degradation system of C. hutchinsonii.
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