Construction and validation of a GFP-based vector for promoter expression analysis in the fish pathogen Flavobacterium psychrophilum

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• 作者：Esther Gó ; mez^a ; David Pé ; rez-Pascual^b ; Lucí ; a Ferná ; ndez^c ; Jessica Mé ; ndez^a ; Pilar Reimundo^a ; Roberto Navais^a ; José ; A. Guijarro^a ; ^{jaga@uniovi.es}
• 关键词：A525 ; absorvance at 525 nm ; ANOVA ; analysis of variance ; cfu ; colony-forming unit ; DNA ; deoxyribonucleic acid ; FRU ; fluorescence relative unit ; GFP ; green fluorescent protein ; MCS ; multiple cloning site ; NA ; nutrient agar ; NAC ; nutrient agar charcoal ; NB
• 刊名：Gene
• 出版年：2012
• 期刊代码：73_03781119
• 类别：bio
• 出版时间：15 April, 2012
• 卷：497
• 期：2
• 页码：263-268
• 文件大小：544 K

摘要

The study of the fish pathogen Flavobacterium psychrophilum has been drastically hampered by the difficulty to perform genetic manipulation of this organism. Although recent publications described the successful transfer of genetic material into this bacterium by transformation and conjugation, additional tools are still needed. This paper reports the construction of vector pCP23-G, which permits for the first time to monitor transcriptional regulation in this pathogen by using a promoterless gfpmut3 gene as a reporter. Additionally, use of pCP23-G enabled the trancriptional analysis of three putative promoter regions of F. psychrophilum, corresponding to genes fpp2-fpp1, pdhB and gldJ, under different growth conditions. Overall, the construction of pCP23-G facilitates genetic analysis in F. psychrophilum, by enabling the determination of gene expression both in vitro and in vivo. Furthermore, this would also open the possibility for studies on the location of this bacterium in the fish tissues.