A family of promoter probe vectors incorporating autofluorescent and chromogenic reporter proteins for studying gene expression in Gram-negative bacteria

• Published in: Microbiology (Society for General Microbiology) 2005-10, Vol.151 (10), p.3249-3256
• Main Authors: Karunakaran, R, Mauchline, T. H, Hosie, A. H. F, Poole, P. S
• Format: Article
• Language: English
• Subjects: Adeno-associated virus; Bacterial Proteins - genetics; Bacterial Proteins - metabolism; Bacteriology; Biological and medical sciences; Dicarboxylic Acid Transporters - genetics; Dicarboxylic Acid Transporters - metabolism; DNA Probes - genetics; Flow Cytometry; Fundamental and applied biological sciences. Psychology; Gene Expression; Genes, Reporter; Genetic Vectors; Genetics; Gram-Negative Bacteria - genetics; Gram-Negative Bacteria - metabolism; Green Fluorescent Proteins - genetics; Green Fluorescent Proteins - metabolism; Luminescent Proteins - genetics; Luminescent Proteins - metabolism; Microbiology; Plasmids; Promoter Regions, Genetic - genetics; Recombinant Fusion Proteins - genetics; Recombinant Fusion Proteins - metabolism; Rhizobium leguminosarum; Rhizobium leguminosarum - enzymology; Rhizobium leguminosarum - genetics; Rhizobium leguminosarum - pathogenicity; Vicia - microbiology
• ISSN: 1350-0872; 1465-2080
• DOI: 10.1099/mic.0.28311-0

School of Biological Sciences, University of Reading, Whiteknights, PO Box 228, Reading RG6 6AJ, UK Correspondence P. S. Poole p.s.poole{at}reading.ac.uk A series of promoter probe vectors for use in Gram-negative bacteria has been made in two broad-host-range vectors, pOT (pBBR replicon) and pJP2 (incP replicon). Reporter fusions can be made to gfpUV , gfpmut3.1 , unstable gfpmut3.1 variants (LAA, LVA, AAV and ASV), gfp +, dsRed2 , dsRedT.3 , dsRedT.4 , mRFP1 , gusA or lacZ . The two vector families, pOT and pJP2, are compatible with one another and share the same polylinker for facile interchange of promoter regions. Vectors based on pJP2 have the advantage of being ultra-stable in the environment due to the presence of the parABCDE genes. As a confirmation of their usefulness, the dicarboxylic acid transport system promoter ( dctA p ) was cloned into a pOT (pRU1097)- and a pJP2 (pRU1156)-based vector and shown to be expressed by Rhizobium leguminosarum in infection threads of vetch. This indicates the presence of dicarboxylates at the earliest stages of nodule formation. Abbreviations: AFP, autofluorescent protein; FAC sorter, fluorescence-activated cell sorter; FACS, fluorescence-activated cell sorting; GFP, green fluorescent protein Present address: Department of Microbiology, The Dental Institute, King's College London, Floor 28, Guy's Tower, Guy's Hospital, London SE1 9RT, UK.