Methods Construction and validation of a GFP-based vector for promoter expression analysis in the fish pathogen Flavobacterium psychrophilum Esther Gómez a , David Pérez-Pascual b , Lucía Fernández c , Jessica Méndez a , Pilar Reimundo a , Roberto Navais a , José A. Guijarro a, ⁎ a Área de Microbiología, Departamento de Biología Funcional, Facultad de Medicina, IUBA, Universidad de Oviedo, 33006 Oviedo, Asturias, Spain b Equipe de Peptides et Communication Bactérienne, Domaine de Vilvert, Institut Micalis, INRA, 78352 Jouy-en-Josas, France c Centre for Microbial Diseases and Immunity Research, Department of Microbiology and Immunology, University of British Columbia, No. 232, 2259 Lower Mall Research Station, Vancouver, British Columbia, Canada V6T 1Z4 abstract article info Article history: Accepted 26 January 2012 Available online 2 February 2012 Keywords: GFP expression vector Flavobacterium psycrophilum Fish pathogen 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 reg- ulation 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 anal- ysis 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. © 2012 Elsevier B.V. All rights reserved. 1. Introduction Flavobacterium psychrophilum belongs to the Cytophaga-Flavobacter- ium-Bacteroides group, which is included in the phylum Bacteroidetes. This bacterium is the etiological agent of cold water disease, a worldwide fish pathology causing important economic losses in salmonid aquacul- ture. In spite of the increasing importance of this infection (Cipriano and Holt, 2005), little is known about the factors that govern the physi- ology and genetics of this pathogen. At present, F. psychrophilum is a bac- terium difficult to manipulate genetically (Álvarez et al., 2004, 2006; Pérez-Pascual et al., 2011). The main reasons for this are that transforma- tion and conjugation frequencies are very low (Álvarez et al., 2004), growth is difficult and slow (Álvarez and Guijarro, 2007) and recovery of the cells on solid media comes up against the presence of viable non-cultivable cells (Michel et al., 1999). Therefore, the knowledge of the virulence factors employed by this microorganism to cause disease is still fairly limited. In the last few years, the development of some ge- netic techniques (Álvarez et al., 2004; Pérez-Pascual et al., 2011), together with the availability of the complete genome sequence of F. psy- chrophilum (Duchaud et al., 2007) have opened new possibilities for the study of virulence determinants in this bacterium. Thus, a few factors as- sociated to pathogenesis have been described (Álvarez et al., 2006, 2008; Bertolini et al., 1994; Crump et al., 2005; Lorenzen and Olesen, 1997; Møller et al., 2003; Nematollahi et al., 2003; Ostland et al., 2000) and pu- tative virulence genes have been identified in the genome analysis of this bacterium (Duchaud et al., 2007). Fluorescence induction with GFP (Green Fluorescent Protein) has been very useful for studying bacterial gene expression as well as inter- actions between the microorganism and the host tissues in several fish pathogens such as Yersinia ruckeri (Welch and Wiens, 2005), Listonella anguillarum (formerly Vibrio anguillarum)(O'Toole et al., 2004) and Edwarsiella tarda (Ling et al., 2000; Ling et al., 2001). A gfp-based report- er system was also developed for the selection of strong promoters in Flavobacterium hibernum and Flavobacterium johnsoniae (Chen et al., 2007, 2010). Additionally, Staroscik et al. (2008) reported the construc- tion of a GFP-based expression vector as a new genetic manipulation tool for Flavobacterium columnare. Thus far, the major problem encoun- tered for the establishment of similar tools in F. psychrophilum was the difficulty of its genetic manipulation. In 2004, Álvarez et al. validated the utilization of β-galactosidase activity as a reporter of gene expres- sion in F. psychrophilum. However, the vector pCP23-β containing the lacZY genes required further modifications so that it could be used for testing promoter sequences of interest; in particular, the insertion of a transcriptional terminator just downstream of the promoter present in the pCP23 plasmid backbone located upstream of the MCS. Moreover, Gene 497 (2012) 263–268 Abbreviations: A525, absorvance at 525 nm; ANOVA, analysis of variance; cfu, col- ony-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, nutient broth; PCR, polymerase chain reaction; ORF, open reading frame; PBS, phosphate buffered saline; rpm, revolutions per minute. ⁎ Corresponding author at: Área de Microbiología, Departamento de Biología Funcional, Facultad de Medicina, Universidad de Oviedo, Asturias, Spain. Tel.: +34 985103000x4218; fax: +34 985103534. E-mail address: jaga@uniovi.es (J.A. Guijarro). 0378-1119/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.gene.2012.01.069 Contents lists available at SciVerse ScienceDirect Gene journal homepage: www.elsevier.com/locate/gene