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
Cited by
45 articles.
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