Strategy for in situ detection of natural transformation-based horizontal gene transfer events

Abstract

A strategy is described that enables the in situ detection of natural transformation in Acinetobacter baylyi BD413 by the expression of a green fluorescent protein. Microscale detection of bacterial transformants growing on plant tissues was shown by fluorescence microscopy and indicated that cultivation-based selection of transformants on antibiotic-containing agar plates underestimates transformation frequencies.

FIG. 1.

(A) Schematic representation (not drawn to scale) of the genetic composition of relevant regions in the donor DNA and in the recipient reporter strain A. baylyi BD413(rbcL-ΔPaadA::gfp). The recipient strain has been designed to recombine with donor DNA containing a functional aadA cassette inserted between the rbcL and the accD genes of the tobacco chloroplast genome. In the donor DNA, the transcription of the aadA gene is under the control of a modified plastid rRNA operon promoter, P_rrn, and the 3′ region of the plastid psbA gene (T_psbA). The reporter strain contains a similar but promoter-free gene fusion, aadA::gfp, in which the aadA domain is physically linked to a gfp domain by an amino acid linker, L. A portion of the rbcL gene has been also cloned upstream from the aadA domain to provide a region for a second crossover event. The absence of a promoter (indicated by a dashed line upstream from the aadA domain) confers a spectomycin-susceptible and GFP-negative phenotype to the reporter strain. The region that is replaced by the double crossover event in the homologous recombination process is drawn in white. Arrows indicate genes. The relevant promoters and terminators are indicated by squares labeled with P and T, respectively. MRT, marker rescue transformation. (B) The expression of the aadA::gfp fusion in the bacterial transformants results in cell fluorescence. Transformant cells were added to a suspension of nontransformed cells of the reporter strain BD413(rbcL-ΔPaadA::gfp) and observed by phase-contrast (left) and fluorescence microscopy (right).

FIG. 2.

Visualization by CLSM of the results of a typical transformation experiment of A. baylyi BD413(rbcL-ΔPaadA::gfp) exposed to cell lysates of E. coli XL1-Blue(pCLT) and incubated for 2 days on a filter membrane. (A) Phase-contrast microscopy image. (B to I) Overlapping (B) of seven 5.8-μm sections (C to I) from the surface (C) to the depth (I) of the bacterial layer. The bar represents 30 μm. The total fluorescence in each layer was quantified by using the Histogram tool of Photoshop 6.0 software. The total green component of each layer was calculated as the average brightness level of the green channel for all the pixels of the image. The green-channel brightness, ranging from 0 to 256 units of brightness, was proportional to the richness in green pixels. The green-channel brightnesses of sections B to I were, respectively, 48.2, 13.2, 11.8, 11.3, 8.2, 4.2, 2.1, and 0.8.

FIG. 3.

Visualization of fluorescent cells and natural transformation of the reporter strain A. baylyi BD413(rbcL-ΔPaadA::gfp) in situ on decaying tobacco tissues. To demonstrate fluorescence, A. baylyi strain BD413(rbcL-aadA::gfp), constitutively expressing GFP, was inoculated onto defrosted leaves and incubated for 5 days. GFP fluorescence showed single cells and microcolonies of A. baylyi in the interstices of epidermal cells (A) and between a stoma and the border of epidermal cells (B). (C to E) In situ detection by CLSM of natural transformation of the reporter strain BD413(rbcL-ΔPaadA::gfp) exposed to externally added DNA on decaying tissues of tobacco. In the experiment whose results are shown in panel C, leaf tissue was supplemented with purified pCLT DNA, while in the experiments whose results are shown in panels D and E, root tissue was supplemented with cell lysates of E. coli XL1-Blue(pCLT). The images show bacterial transformants on a leaf surface near a stoma (C) or scattered along the root tissues (D and E). Bars represent 10 μm.