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
Jazyk angličtina Země Spojené státy americké Médium print-electronic
Typ dokumentu časopisecké články, práce podpořená grantem
- MeSH
- Bacillus genetika MeSH
- bakteriální transformace * MeSH
- barvení a značení MeSH
- elektroporace MeSH
- genetické vektory MeSH
- inzerční mutageneze metody MeSH
- kultivační média chemie MeSH
- průtoková cytometrie MeSH
- rýže (rod) mikrobiologie MeSH
- transpozibilní elementy DNA MeSH
- zelené fluorescenční proteiny genetika metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- kultivační média MeSH
- transpozibilní elementy DNA MeSH
- zelené fluorescenční proteiny MeSH
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.
Zobrazit více v PubMed
Appl Environ Microbiol. 1989 Mar;55(3):604-10 PubMed
BMC Microbiol. 2011 May 16;11:107 PubMed
J Bacteriol. 1990 Nov;172(11):6568-72 PubMed
Annu Rev Microbiol. 2009;63:541-56 PubMed
J Microbiol Methods. 2011 Jan;84(1):114-20 PubMed
Curr Microbiol. 2008 Sep;57(3):245-50 PubMed
J Appl Microbiol. 2010 Aug;109(2):657-666 PubMed
BMC Biotechnol. 2007 Mar 21;7:15 PubMed
Proc Natl Acad Sci U S A. 2007 Jan 16;104(3):1009-14 PubMed
Curr Opin Microbiol. 2003 Jun;6(3):310-6 PubMed
Microbiology (Reading). 2001 Sep;147(Pt 9):2529-2536 PubMed
Appl Microbiol Biotechnol. 2010 Feb;85(5):1301-13 PubMed
Nat Biotechnol. 2000 Jan;18(1):97-100 PubMed
Mol Biol Rep. 2010 Jun;37(5):2207-13 PubMed
Anal Biochem. 2011 Feb 1;409(1):130-7 PubMed
Proc Natl Acad Sci U S A. 1983 Apr;80(8):2305-9 PubMed
Biotechnol Lett. 2006 Jul;28(13):979-85 PubMed
Annu Rev Phytopathol. 2002;40:411-41 PubMed
J Med Microbiol. 2003 Jan;52(Pt 1):69-74 PubMed
Curr Microbiol. 2004 Dec;49(6):428-32 PubMed
Appl Microbiol Biotechnol. 2008 Oct;80(5):937-44 PubMed
Mol Plant Microbe Interact. 2000 Sep;13(9):951-61 PubMed
J Environ Sci Health B. 2009 May;44(4):397-402 PubMed
Mol Plant. 2010 Jan;3(1):246-59 PubMed
Curr Microbiol. 2001 Oct;43(4):244-8 PubMed
Appl Microbiol Biotechnol. 2008 Apr;78(6):1033-43 PubMed
Gene. 1993 Jan 15;123(1):17-24 PubMed
Nucleic Acids Res. 2005 Oct 12;33(18):e153 PubMed
Folia Microbiol (Praha). 2005;50(5):437-42 PubMed
