title: FLP-FRT-based method to obtain unmarked deletions of CHU_3237 (porU) and large genomic fragments of Cytophaga hutchinsonii.
abstract: Cytophaga hutchinsonii is a widely distributed cellulolytic bacterium in the phylum Bacteroidetes. It can digest crystalline cellulose rapidly without free cellulases or cellulosomes. The mechanism of its cellulose utilization remains a mystery. We developed an efficient method based on a linear DNA double-crossover and FLP-FRT recombination system to obtain unmarked deletions of both single genes and large genomic fragments in C. hutchinsonii. Unmarked deletion of CHU_3237 (porU), an ortholog of the C-terminal signal peptidase of a type IX secretion system (T9SS), resulted in defects in colony spreading, cellulose degradation, and protein secretion, indicating that it is a component of the T9SS and that T9SS plays an important role in cellulose degradation by C. hutchinsonii. Furthermore, deletions of four large genomic fragments were obtained using our method, and the sizes of the excised fragments varied from 9 to 19 kb, spanning from 6 to 22 genes. The customized FLP-FRT method provides an efficient tool for more rapid progress in the cellulose degradation mechanism and other physiological aspects of C. hutchinsonii.
authors: State Key Laboratory of Microbial Technology, School of Life Science, Shandong University, Jinan, China.
authors: Ying
authors: Y
authors: Wang
authors: State Key Laboratory of Microbial Technology, School of Life Science, Shandong University, Jinan, China.
authors: Zhiquan
authors: Z
authors: Wang
authors: State Key Laboratory of Microbial Technology, School of Life Science, Shandong University, Jinan, China.
authors: Jing
authors: J
authors: Cao
authors: State Key Laboratory of Microbial Technology, School of Life Science, Shandong University, Jinan, China.
authors: Zhiwei
authors: Z
authors: Guan
authors: State Key Laboratory of Microbial Technology, School of Life Science, Shandong University, Jinan, China luxuemei@sdu.edu.cn.
authors: Xuemei
authors: X
authors: Lu
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D002482:Cellulose / Q000378:metabolism; D003434:Crossing Over, Genetic; D003589:Cytophaga / Q000235:genetics* / Q000502:physiology; D018390:Gene Targeting / Q000379:methods*; D005818:Genetic Engineering / Q000379:methods*; D016680:Genome, Bacterial / Q000235:genetics*; D023281:Genomics; D010641:Phenotype; D045522:Recombinases / Q000235:genetics / Q000378:metabolism; D017384:Sequence Deletion
chemical_list: D001426:Bacterial Proteins; D045522:Recombinases; D002482:Cellulose