|ZFIN ID: ZDB-PUB-100408-24|
Broad-host-range Plasmids for Red Fluorescent Protein Labeling of Gram-negative Bacteria for Use in the Zebrafish Model System
Singer, J.T., Phennicie, R.T., Sullivan, M.J., Porter, L.A., Shaffer, V.J., and Kim, C.H.
|Source:||Applied and environmental microbiology 76(11): 3467-3474 (Journal)|
|Registered Authors:||Kim, Carol H.|
|PubMed:||20363780 Full text @ Appl. Environ. Microbiol.|
Singer, J.T., Phennicie, R.T., Sullivan, M.J., Porter, L.A., Shaffer, V.J., and Kim, C.H. (2010) Broad-host-range Plasmids for Red Fluorescent Protein Labeling of Gram-negative Bacteria for Use in the Zebrafish Model System. Applied and environmental microbiology. 76(11):3467-3474.
ABSTRACTIn order 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 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 non-toxic and broadly transmissible to a variety of Gram-negative bacteria. DNA fragments encoding the RFP 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 E. 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 was likely due to a mutation in lacI(q) carried on pMMB66EH. DNA sequence analysis confirmed the presence of 5 transitions, 4 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 studies of real-time interactions between host cells of the innate immune system and the infecting pathogen.