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Development of a high-efficient transformation system of Bacillus pumilus strain DX01 to facilitate gene isolation via gfp-tagged insertional mutagenesis and visualize bacterial colonization of rice roots.
MedLine Citation:
PMID:  23334949     Owner:  NLM     Status:  Publisher    
Abstract/OtherAbstract:
A Tn5 transposition vector, pMOD-tet-egfp, was constructed and used for the random insertional mutagenesis of Bacillus pumilus. Various parameters were investigated to increase the transformation efficiency B. pumilus DX01 via Tn5 transposition complexes (transposome): bacterial growth phase, type of electroporation buffer, electric field strength, and recovery medium. Transformation efficiency was up to 3 × 10(4) transformants/μg of DNA under the optimized electroporation conditions, and a total of 1,467 gfp-tagged transformants were obtained. Fluorescence-activated cell sorting analysis showed that all gfp-tagged bacterial cells expressed GFP, indicating that foreign DNA has been successfully integrated into the genome of B. pumilus and expressed. Finally, flanking DNA sequences were isolated from several transformants and colonization of rice roots by B. pumilus DX01 was also studied. The method developed here will be useful for creating an insertion mutant library of gram-positive bacteria, thus facilitating their molecular genetic and cytological studies.
Authors:
Xinqian Shen; Yunpeng Chen; Tong Liu; Xiaolu Hu; Zhenfang Gu
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Publication Detail:
Type:  JOURNAL ARTICLE     Date:  2013-1-22
Journal Detail:
Title:  Folia microbiologica     Volume:  -     ISSN:  1874-9356     ISO Abbreviation:  Folia Microbiol. (Praha)     Publication Date:  2013 Jan 
Date Detail:
Created Date:  2013-1-21     Completed Date:  -     Revised Date:  -    
Medline Journal Info:
Nlm Unique ID:  0376757     Medline TA:  Folia Microbiol (Praha)     Country:  -    
Other Details:
Languages:  ENG     Pagination:  -     Citation Subset:  -    
Affiliation:
Department of Resources and Environmental Sciences, School of Agriculture and Biology, Shanghai Jiaotong University, Shanghai, 200240, People's Republic of China.
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