Open AccessA bifunctional transposon mini‐Tn 5gfp‐km which can be used to select for promoter fusions and report gene expression levels in Agrobacterium tumefaciens
Author(s) -
Tang Xie,
Lu Bai F,
Pan Shen Q
Publication year - 1999
Publication title -
fems microbiology letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.899
H-Index - 151
eISSN - 1574-6968
pISSN - 0378-1097
DOI - 10.1111/j.1574-6968.1999.tb08704.x
Subject(s) - agrobacterium tumefaciens , transposable element , promoter , biology , acetosyringone , mutant , reporter gene , green fluorescent protein , kanamycin , microbiology and biotechnology , operon , gene , genetics , gene expression , transformation (genetics)
A mini‐Tn 5 transposon derivative, mini‐Tn 5gfp‐km , has been constructed which contained a promoter‐less artificial operon consisting of two open reading frames, green fluorescent protein (GFP) and neomycin phosphotransferase II (NptII). When this transposon was used to mutagenize Agrobacterium tumefaciens , all the mutants selected in the presence of kanamycin exhibited GFP expression, which could be conveniently monitored by a fluorometer. The transposon appeared to be bifunctional and could provide both selection and reporter functions. Even the mutants showing minimal levels of GFP expression were still resistant to kanamycin. This suggests that this transposon can be used to select for insertions downstream of both weak and strong promoters, as long as the insertions themselves are non‐lethal. This system was used to identify A. tumefaciens genes that were upregulated in response to acidic pH. Screening only 20 colonies led to identification of two promoters that were specifically induced by low pH and one promoter that was specifically induced by acetosyringone in a minimal medium of pH 5.5.