Article Figures & Data
Figures
- FIG. 1.
General structure of pigment-coding regions in pRSETB plasmids carrying d-Tomato, td-Tomato, m-Orange, and m-Cherry DNAs. All pigment DNAs were carried on XbaI-HindIII fragments, and structural pigment sequences could be isolated only on smaller BamHI-HindIII fragments. Sequences upstream of the ATG start site for pigment production contained a ribosome binding site, 6-His codons, and a 62-bp region encoding an antigenic epitope for immunological detection (25).
- FIG. 2.
E. coli HB101 strains carrying IPTG-inducible pMMB66EH derivatives expressing RFPs. Strains were streaked on L agar plates containing ampicillin. The plate on the right contained 0.4 mM IPTG. Plasmids carried by each strain are the following (clockwise from the top): p66MC1 (m-Cherry), p66M1 (m-Orange), p66TD1 (td-Tomato), and p66T1 (d-Tomato).
- FIG. 3.
P. aeruginosa PA01(p66TDC1) and PA14(p66TDC1) strains carrying a constitutive variant of p66TD1. Strains were streaked on L agar plates containing ampicillin only. The plate on the left shows pigment production by PA01(p66TDC1), and that on the right shows pigment production by PA14(p66TDC1).
- FIG. 4.
DNA sequence of lacIq carried by plasmids pMMB66EH and p66TDC1. A 1,220-bp region containing the lacI genes of pMMB66EH and p66TDC1 was subjected to automated DNA sequencing. A portion of the sequence from nucleotide (nt) 724 to 771 is shown above. Numbers above the sequence denote nucleotide positions. Letters above and below the sequences indicate amino acid assignments. Single-base-pair substitutions are indicated by an asterisk. The 2-bp insertion at nt 742 in p66TDC1 lacI is underlined.
- FIG. 5.
E. coli HB101 (lacI+) and P. aeruginosa PA14 (lacI−) carrying constitutive p67 derivatives expressing RFPs. Strains were streaked on L agar plates containing ampicillin. E. coli HB101 is on the left and P. aeruginosa PA14 is on the right. Plasmids carried by each strain are the following (clockwise from the top): p67MC1 (m-Cherry), p67M1 (m-Orange), p67TD1 (td-Tomato), and p67T1 (d-Tomato).
- FIG. 6.
Infection of zebrafish embryos (48 hpf) with P. aeruginosa PA14(p67T1) encoding d-Tomato. Embryos were microinjected with 50 CFU of P. aeruginosa PA14(p67T1) through the duct of Cuvier and imaged at 6 hpi. (A) Image analysis using an Olympus IX-81 inverted epifluorescence microscope equipped with a Hammatsu ORCA-ER CCD camera and IPLab software. Red fluorescence was observable at ×40 magnification (shown) or higher. Punctate fluorescence was observed in many tissues, including the vasculature, the yolk, and the pericardial sac. The image is an overlay of wide-field differential interference contrast (DIC) and fluorescence images. (B and C) Image analysis using an Olympus IX-81 and FV-1000 laser-scanning confocal microscope with Olympus FV-10 software. (B) Tg(fli1::EGFP) zebrafish express EGFP in macrophages (15) and were observed to contain phagocytosed P. aeruginosa PA14(p67T1) encoding d-Tomato. Three green fluorescent macrophages along the peripheral yolk are shown in association at ×1,000 magnification, with one containing RFP-labeled P. aeruginosa PA14(p67T1). (C) Tg(mpo::GFP) zebrafish express GFP in neutrophils (22) and were observed containing phagocytosed P. aeruginosa PA14(p67T1) encoding d-Tomato. The figure is at ×1,000 magnification. Filters providing excitation/emission at 543 nm/610 nm and 488 nm/510 nm were used for the detection of RFP and EGFP, respectively.
Tables
- TABLE 1.
Bacterial strains and plasmids
Name Relevant characteristic(s) Source or reference(s) Bacterial strains Escherichia coli HB101 Rifs; cloning host and conjugal donor 3 Edwardsiella tarda Ap MIC, <100 μg/ml 21 Edwardsiella tarda ETR1 Spontaneous Rifr mutant of E. tarda, conjugal recipient for RFP plasmids This work Pseudomonas aeruginosa PA01 Ap MIC, 1,050 μg/ml George O'Toole, Dartmouth Medical School Pseudomonas aeruginosa PA01R1 Spontaneous Rifr mutant of PAO1, conjugal recipient for RFP plasmids This work Pseudomonas aeruginosa PA14 Ap MIC, 600 μg/ml; Rifr conjugal recipient for RFP plasmids This work Vibrio (Listonella) anguillarum 775(pJM1) Serotype O1 marine fish pathogen; Ap MIC, 200 μg/ml 7 Vibrio (Listonella) anguillarum 775R1(pJM1) Spontaneous Rifr mutant of 775(pJM1), conjugal recipient for RFP plasmids 26 Vibrio (Listonella) anguillarum H775-3 pJM1-cured derivative of 775(pJM1) 6 Vibrio (Listonella) anguillarum H775-3R1 Spontaneous Rifr mutant of H775-3, conjugal recipient for RFP plasmids 26 Vibrio (Listonella) anguillarum NVI5812 Serotype O2β cod pathogen; Ap MIC, 400 μg/ml Una McCarthy, University of Maine Vibrio (Listonella) anguillarum NVI5812R1 Spontaneous Rifr mutant of NVI5812, conjugal recipient for RFP plasmids This work Plasmids pMMB66EH 8.8-kb Apr moderate-copy IncQ-based vector, mobilizable, lacIq+, IPTG-inducible ptac 1, 12 pUC4-K Source of 1.5-kb BamHI kan DNA fragment 31 p66-Km1 Carries 1.5-kb BamHI kan fragment from pUC4-K cloned into pMMB66EH; Apr Kmr, lacIq+ This work pRK2013 20-kb Kmr ColE1-based restricted-host-range helper plasmid, mobilizes pMMB66EH derivatives 9, 11 pRSETB d-Tomato Source of 852-bp XbaI-HindIII d-Tomato DNA 25 pRSETB td-Tomato Source of 1,578-bp XbaI-HindIII td-Tomato DNA 25 pRSETB m-Orange Source of 858-bp XbaI-HindIII m-Orange DNA 25 pRSETB m-Cherry Source of 848-bp XbaI-HindIII m-Cherry DNA 25 p66T1 pMMB66EH-based d-Tomato construct; Apr; encodes IPTG-inducible pigment production This work p66TD1 pMMB66EH-based td-Tomato construct; Apr; encodes IPTG-inducible pigment production This work p66M1 pMMB66EH-based m-Orange construct; Apr; encodes IPTG-inducible pigment production This work p66MC1 pMMB66EH-based m-Cherry construct, Apr; encodes IPTG-inducible pigment production This work p66TDC1 pMMB66EH-based td-Tomato construct; Apr; carries spontaneous lacI mutations resulting in constitutive pigment production This work p67T1 p66TDC1 BamHI-HindIII lacI− vector fragment carrying 718-bp BamHI-HindIII d-Tomato DNA; Apr; constitutive pigment production This work p67TD1 Like p67T1, but carrying 1,444-bp BamHI-HindIII td-Tomato DNA This work p67M1 Like p67T1, but carrying 724-bp BamHI-HindIII m-Orange DNA This work p67MC1 Like p67T1, but carrying 714-bp BamHI-HindIII m-Cherry DNA This work - TABLE 2.
Complementation of d-Tomato pigment production directed by p66TDC1
Strain Pigment −IPTG +IPTG HB101(p66T1) Colorless Pink HB101(p66T1, p66-kan) Colorless Light pink HB101(p66TDC1) Pink Deep pink HB101(p66TDC1, p66-kan) Colorless Pink - TABLE 3.
Stability of RFP-expressing plasmids under nonselective conditions
Host strain Plasmid loss (%) from overnight cultures p66T1 p66TD1 p67T1 p67M1 p67MC1 EtR1 1 0 49 25 0 PA14 5 97 19 15 10 775R1 0 1 1 1 3 NVI5812R1 5 2 6 2 2
