High rates of conjugation in bacterial biofilms as determined by quantitative in situ analysis

Abstract

Quantitative in situ determination of conjugative gene transfer in defined bacterial biofilms using automated confocal laser scanning microscopy followed by three-dimensional analysis of cellular biovolumes revealed conjugation rates 1,000-fold higher than those determined by classical plating techniques. Conjugation events were not affected by nutrient concentration alone but were influenced by time and biofilm structure.

FIG. 1

Transconjugant cells in a biofilm of A. eutrophus AE104 grown under nutrient-rich (A) and nutrient-poor (B) conditions. Signals were collected consecutively and stored as grey images. With computer-assisted coloring, recipient AE104 cells were assigned the color red. Donor E. coli GM16 cells are depicted in green. Transconjugants emitted both green light (due to GFP) and red light (due to hybridization with the TRITC-labelled rRNA-directed oligonucleotide probe) and are shown in yellow. Bar, 25 μm.

FIG. 2

Average distribution of recipient, donor, and transconjugant cells at different depths in a 7.3 × 10⁴ μm³ volume of biofilm grown in rich medium for 24 h.