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

• Published in: Gene Vol. 497; no. 2; pp. 263 - 268
• Main Authors: Gómez, Esther, Pérez-Pascual, David, Fernández, Lucía, Méndez, Jessica, Reimundo, Pilar, Navais, Roberto, Guijarro, José A.
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 15.04.2012; Elsevier
• Subjects: Animals; bacteria; Escherichia coli - genetics; fish; Fish Diseases - microbiology; Fish pathogen; Fishes - microbiology; Flavobacteriaceae Infections - genetics; Flavobacterium - genetics; Flavobacterium psychrophilum; Flavobacterium psycrophilum; Gene Expression; genetic engineering; Genetic Techniques; genetic techniques and protocols; Genetic Vectors - genetics; GFP expression vector; Green Fluorescent Proteins - genetics; Life Sciences; pathogens; Plasmids - genetics; promoter regions; Promoter Regions, Genetic; reporter genes; transcription (genetics); PBS; ORF; FRU; A525; GFP expression vector; rpm; GFP; NAC; cfu; NA; NB; ANOVA; DNA; Flavobacterium psycrophilum; MCS; PCR; Fish pathogen; SYSTEM; RAINBOW-TROUT; METALLOPROTEASE; ONCORHYNCHUS-MYKISS; VIRULENCE; EDWARDSIELLA-TARDA; PURIFICATION; DISEASE; GLIDING MOTILITY; FLUORESCENT PROTEIN GFP
• ISSN: 0378-1119; 1879-0038
• DOI: 10.1016/j.gene.2012.01.069

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. ► The pCP23-G plasmid was constructed for transcriptional analysis in F. psychrophilum. ► The pCP23-βpth vector was useful for transcriptional analysis in F. psychrophilum. ► GFP fluorescence emission was analyzed by confocal microscopy and flow cytometry. ► fpp2–fpp1 promoter was calcium- and temperature-regulated.