bifunctional transposon mini-Tn5gfp-km which can be used to select for promoter fusions and report gene expression levels in Agrobacterium tumefaciens

• Published in: FEMS microbiology letters Vol. 179; no. 1; pp. 37 - 42
• Main Authors: Tang, X, Lu, B.F, Pan, S.Q
• Format: Journal Article
• Language: English
• Published: Oxford, UK Blackwell Publishing Ltd 01.10.1999; Blackwell; Oxford University Press
• Subjects: Acetosyringone; Agrobacterium tumefaciens; Agrobacterium tumefaciens - genetics; Artificial operon; Biological and medical sciences; Biotechnology; Cloning, Molecular; DNA Transposable Elements; Escherichia coli - genetics; Fluorescence; Fluorometers; Fundamental and applied biological sciences. Psychology; Gene expression; Genes, Reporter; Genetic engineering; Genetic technics; gfp; Green fluorescent protein; Green Fluorescent Proteins; Kanamycin; Kanamycin Kinase - biosynthesis; Kanamycin Kinase - genetics; Luminescent Proteins - biosynthesis; Luminescent Proteins - genetics; Methods. Procedures. Technologies; Microbiology; Neomycin; Neomycin phosphotransferase; nptII; Open Reading Frames; Operon; pH effects; Phosphotransferase; Phosphotransferase II; Promoter Regions, Genetic; Promoters; Recombinant Fusion Proteins - biosynthesis; Selection‐reporter transposon; Transcriptional fusion; Transposons; Vectors (cloning, transfer, expression). Insertion sequences and transposons; Selection-reporter transposon; Transcriptional fusion; Artificial operon; Regulation(control); Reporter gene; Transcription promoter; Insertion mutation; Selection; Transposon; Agrobacterium tumefaciens; Biological marker; Bacteria; Rhizobiaceae; Transposable element
• ISSN: 0378-1097; 1574-6968
• DOI: 10.1111/j.1574-6968.1999.tb08704.x

A mini-Tn5 transposon derivative, mini-Tn5gfp-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 trasposon 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.