title: Broad-host-range plasmids for red fluorescent protein labeling of gram-negative bacteria for use in the zebrafish model system.
abstract: To observe real-time interactions between green fluorescent protein-labeled immune cells and invading bacteria in the zebrafish (Danio rerio), a series of plasmids was constructed for the red fluorescent protein (RFP) labeling of a variety of fish and human pathogens. The aim of this study was to create a collection of plasmids that would express RFP pigments both constitutively and under tac promoter regulation and that would be nontoxic and broadly transmissible to a variety of Gram-negative bacteria. DNA fragments encoding the RFP dimeric (d), monomeric (m), and tandem dimeric (td) derivatives d-Tomato, td-Tomato, m-Orange, and m-Cherry were cloned into the IncQ-based vector pMMB66EH in Escherichia coli. Plasmids were mobilized into recipient strains by conjugal mating. Pigment production was inducible in Escherichia coli, Pseudomonas aeruginosa, Edwardsiella tarda, and Vibrio (Listonella) anguillarum strains by isopropyl-beta-d-thiogalactopyranoside (IPTG) treatment. A spontaneous mutant exconjugant of P. aeruginosa PA14 was isolated that expressed td-Tomato constitutively. Complementation analysis revealed that the constitutive phenotype likely was due to a mutation in lacI(q) carried on pMMB66EH. DNA sequence analysis confirmed the presence of five transitions, four transversions, and a 2-bp addition within a 14-bp region of lacI. Vector DNA was purified from this constitutive mutant, and structural DNA sequences for RFP pigments were cloned into the constitutive vector. Exconjugants of P. aeruginosa, E. tarda, and V. anguillarum expressed all pigments in an IPTG-independent fashion. Results from zebrafish infectivity studies indicate that RFP-labeled pathogens will be useful for the study of real-time interactions between host cells of the innate immune system and the infecting pathogen.
authors.affiliation: Department of Molecular and Biomedical Sciences, University of Maine, Orono, ME 04469, USA. jsinger@maine.edu
authors.forename: John T
authors.initials: JT
authors.lastname: Singer
authors.forename: Ryan T
authors.initials: RT
authors.lastname: Phennicie
authors.forename: Matthew J
authors.initials: MJ
authors.lastname: Sullivan
authors.forename: Laura A
authors.initials: LA
authors.lastname: Porter
authors.forename: Valerie J
authors.initials: VJ
authors.lastname: Shaffer
authors.forename: Carol H
authors.initials: CH
authors.lastname: Kim
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D004195:Disease Models, Animal; D005822:Genetic Vectors; D006090:Gram-Negative Bacteria / Q000235:genetics / Q000472:pathogenicity*; D054884:Host-Pathogen Interactions; D008164:Luminescent Proteins / Q000235:genetics* / Q000378:metabolism*; D008967:Molecular Biology / Q000379:methods*; D008969:Molecular Sequence Data; D010957:Plasmids; D017422:Sequence Analysis, DNA; D013194:Staining and Labeling / Q000379:methods*; D015027:Zebrafish / Q000382:microbiology; D000097573:Red Fluorescent Protein
chemical_list: D004269:DNA, Bacterial; D008164:Luminescent Proteins; D000097573:Red Fluorescent Protein