title: Development of replicative oriC plasmids and their versatile use in genetic manipulation of Cytophaga hutchinsonii.
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.
authors.affiliation: State Key Laboratory of Microbial Technology, College of Life Science, Shandong University, Jinan, 250100, China.
authors.forename: Yuanxi
authors.initials: Y
authors.lastname: Xu
authors.forename: Xiaofei
authors.initials: X
authors.lastname: Ji
authors.forename: Ning
authors.initials: N
authors.lastname: Chen
authors.forename: Pengwei
authors.initials: P
authors.lastname: Li
authors.forename: Weifeng
authors.initials: W
authors.lastname: Liu
authors.forename: Xuemei
authors.initials: X
authors.lastname: Lu
mesh_terms: D003589:Cytophaga / Q000235:genetics*; D018274:Electroporation / Q000379:methods; D004926:Escherichia coli / Q000235:genetics; D005818:Genetic Engineering / Q000379:methods; D005822:Genetic Vectors; D005827:Genetics, Microbial / Q000379:methods; D008967:Molecular Biology / Q000379:methods; D010957:Plasmids; D018741:Replication Origin; D014169:Transformation, Bacterial