Stable, site-specific fluorescent tagging constructs optimized for burkholderia species

Abstract

Several vectors that facilitate stable fluorescent labeling of Burkholderia pseudomallei and Burkholderia thailandensis were constructed. These vectors combined the effectiveness of the mini-Tn7 site-specific transposition system with fluorescent proteins optimized for Burkholderia spp., enabling bacterial tracking during cellular infection.

FIG. 1.

Maps of mini-Tn7-gat-cfp and mini-Tn7-kan-cfp (A), mini-Tn7-gat-gfp and mini-Tn7-kan-gfp (B), mini-Tn7-gat-rfp and mini-Tn7-kan-rfp (C), and mini-Tn7-gat-yfp and mini-Tn7-kan-yfp (D). These constructs allow for site-specific transposition of fluorescent protein genes (cfp, gfp, rfp, and yfp), using the nonantibiotic glyphosate resistance marker (gat) or the kanamycin resistance marker (kan) assisted by the helper plasmid pTNS3-asd_Ec. Differences in plasmid size are indicated in parenthesis. The P_S12 promoter drives all fluorescent proteins. The gat or kan cassette is driven by the rpsL promoter of B. cenocepacia (PC_S12) on all constructs. These selectable markers are flanked by FRT sequences for Flp protein-mediated excision. Abbreviations: oriT, RP4 conjugal origin of transfer; PC_S12, rpsL promoter of B. cenocepacia; P_S12, rpsL promoter of B. pseudomallei; R6K_γori, π protein-dependent R6K origin of replication (γ indicates subtype of the origin); Tn7L and Tn7R, left and right transposase recognition sequences; T₀T₁, transcriptional terminator.

FIG. 2.

Fluorescence microscopy of B. pseudomallei labeled at the att-Tn7 site with gat-cfp (A), gat-gfp (B), gat-rfp (C), and gat-yfp (D). Fluorescent signals were obtained using the respective filter cube sets on a Zeiss AxioObserver D1 microscope and AxioCam MRc 5 monochrome camera. Pseudocolor was applied to the signal intensity at the time of capture using Zeiss AxioVision software. The middle row is comprised of differential interference contrast (DIC) images from the respective samples. At the time of capture, Zeiss AxioVision software was used to superimpose the fluorescent signal and DIC images, displayed in the bottom row. When comparing the overlay in the bottom row to the DIC image in the middle, it can be seen that almost all bacteria are fluorescing at one exposure time or at a set fluorescent intensity. Differing levels of expression of fluorescent protein genes and extended fixation can result in slightly different fluorescent intensities among a population of bacteria, since extended paraformaldehyde fixation could damage the fluorescent proteins (12). The total magnification is ×630, and scale bars equal 10 μm.

FIG. 3.

Fluorescence microscopy of B. pseudomallei labeled at the att-Tn7 site with gat-cfp (A), gat-gfp (B), gat-rfp (C), and gat-yfp (D) to infect the murine macrophage-like cell line RAW 264.7. Cell monolayers were seeded overnight onto poly-l-lysine-coated coverslips at the bottom of a 6-well plate and infected with fluorescently tagged B. pseudomallei. Images were obtained as described in the legend of Fig. 2. The total magnification is ×630, and scale bars equal 10 μm.

FIG. 4.

Dual infection of RAW 264.7 macrophages by differentially labeled (green and red) B. pseudomallei bacteria. Infections were carried out identically to those described in the legend of Fig. 3. (A) The green fluorescent signal indicates where gfp-tagged B. pseudomallei bacteria are replicating inside macrophages. (B) The red fluorescent signal was obtained from the same field and shows where rfp-tagged B. pseudomallei bacteria are replicating within macrophages. (C) A DIC image was then captured and is presented. (D) Overlay of images captured sequentially in panels A, B, and C. Images were superimposed at the time of capture using Zeiss AxioVision software. (E and F) Close-ups of the two macrophages indicated by arrows in panel D, where the two differently fluorescing B. pseudomallei strains are clearly visible and distinguishable within the macrophages and even within the same host cell. The total magnification in panels A to D is ×630, and all scale bars equal 10 μm.

FIG. 5.

Tracking of B. pseudomallei infectious stages. Infections were carried out as described in the legend of Fig. 3 except that B. pseudomallei bacteria were used to infect macrophages at an MOI of 1:5 to enable isolated bacterial infection. (A) RAW 264.7 monolayers were infected with RFP-tagged B. pseudomallei. The infection was allowed to progress for 1 h, and then vesicles were stained far-red with the lipophilic styryl dye FM-4-64-FX (Molecular Probes). Phase-contrast microscopy in the red fluorescent channel captured an image of two RFP-tagged B. pseudomallei bacteria in a phagocytic vesicle. The image in panel B was obtained similarly, except that a single RFP-tagged B. pseudomallei bacterium is possibly escaping the far-red-stained phagocytic vesicle. (C) RAW 264.7 macrophages were infected with GFP-tagged B. pseudomallei for 2 h, after which the monolayers were fixed and permeabilized and host cell actin was stained far-red with phalloidin (Invitrogen). GFP-tagged B. pseudomallei can be seen polymerizing host cell actin, enabling observation of actin-based intracellular motility. (D) GFP-tagged B. pseudomallei bacteria were used to infect RAW 264.7 monolayers for 6 h. The bacteria are polymerizing host cell actin to infect neighboring host cells via membrane protrusions. The arrows indicate GFP-tagged B. pseudomallei bacteria at the tips of polymerized actin tails. The total magnification in all panels is ×1,000.