Knockout of the adp gene related with colonization in Bacillus nematocida B16 using customized transcription activator-like effectors nucleases.
Bacillus nematocida B16 is able to dominate in the intestines of the worm Caenorhabditis elegans in 'Trojan horse' pathogenic mechanism. The adp is one candidate gene which potentially play a vital role in the colonization from our previous random mutagenesis screening results. To analyse the functional role of this gene, we constructed the adp knockout mutant through customized transcription activator-like effectors nucleases (TALEN), which has been successfully used in yeasts, nematodes, zebrafish and human pluripotent cells. Here, we first time report this knockout method in bacteria on this paper. Bioassay experiments demonstrated that the adp knockout mutant of B16 showed considerably lower colonization activity, reduced numbers of intestines and less than 80% nematocidal activity compared with the wild-type strain when infected for 48 h. However, no obvious change on proteolytic activity was observed in the mutant. Conversely, the complementation of adp gene restored most of the above deficient phenotypes. These results indicated that the adp gene was involved in surface adhesion and played a comparatively important role in colonizing host nematodes. Moreover, TALENs successfully disrupt target genes in bacteria.
Department of Life Science and Biotechnology, Nanyang Normal University, Nanyang, Henan, 473061, China.|Laboratory for Conservation and Utilization of Bio-Resources, Key Laboratory for Microbial Resources of the Ministry of Education, Yunnan University, Kunming, 650091, China.
Qiuhong
Q
Niu
Department of Life Science and Biotechnology, Nanyang Normal University, Nanyang, Henan, 473061, China.
Haoying
H
Zheng
Department of Life Science and Biotechnology, Nanyang Normal University, Nanyang, Henan, 473061, China.
Lin
L
Zhang
Department of Pathology, School of Medicine, University of Virginia, Charlottesville, VA, 22908, USA.
Fujun
F
Qin
Department of Pathology, School of Medicine, University of Virginia, Charlottesville, VA, 22908, USA.
Loryn
L
Facemire
Department of Life Science and Biotechnology, Nanyang Normal University, Nanyang, Henan, 473061, China.
Guo
G
Zhang
Department of Life Science and Biotechnology, Nanyang Normal University, Nanyang, Henan, 473061, China.
Feng
F
Cao
Laboratory for Conservation and Utilization of Bio-Resources, Key Laboratory for Microbial Resources of the Ministry of Education, Yunnan University, Kunming, 650091, China.
Ke-Qin
KQ
Zhang
Laboratory for Conservation and Utilization of Bio-Resources, Key Laboratory for Microbial Resources of the Ministry of Education, Yunnan University, Kunming, 650091, China.
Xiaowei
X
Huang
Department of Life Science and Biotechnology, Nanyang Normal University, Nanyang, Henan, 473061, China.
Jianwei
J
Yang
Department of Life Science and Biotechnology, Nanyang Normal University, Nanyang, Henan, 473061, China.
Lei
L
He
Department of Life Science and Biotechnology, Nanyang Normal University, Nanyang, Henan, 473061, China.
Chanjuan
C
Liu
D018829:Adhesins, Bacterial / Q000235:genetics* / Q000378:metabolism; D000818:Animals; D001407:Bacillus / Q000235:genetics* / Q000254:growth & development / Q000472:pathogenicity; D017173:Caenorhabditis elegans / Q000382:microbiology* / Q000502:physiology; D041981:Gastrointestinal Tract / Q000382:microbiology; D055786:Gene Knockout Techniques / Q000379:methods*; D005816:Genetic Complementation Test; D016019:Survival Analysis; D037521:Virulence Factors / Q000235:genetics / Q000378:metabolism*
D018829:Adhesins, Bacterial; D037521:Virulence Factors