Broad-Host-Range Plasmids for Red Fluorescent Protein Labeling of Gram-Negative Bacteria for Use in the Zebrafish Model System

Author:

Singer John T.1,Phennicie Ryan T.1,Sullivan Matthew J.1,Porter Laura A.1,Shaffer Valerie J.1,Kim Carol H.12

Affiliation:

1. Department of Molecular and Biomedical Sciences, 5735 Hitchner Hall, University of Maine, Orono, Maine 04469

2. Graduate School for Biomedical Sciences, ESRB/Barrows Hall, University of Maine, Orono, Maine 04469

Abstract

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-β- 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.

Publisher

American Society for Microbiology

Subject

Ecology,Applied Microbiology and Biotechnology,Food Science,Biotechnology

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