Protocols for Visualizing Horizontal Gene Transfer in Gram-Negative Bacteria Through Natural Competence

Horizontal gene transfer (HGT) is widespread in bacteria and archaea and plays a significant role in prokaryotic evolution. One mode of HGT is natural competence for genetic transformation, which allows the organism to take up free DNA from its surroundings and incorporate this DNA into its own genome. Natural transformation has been studied in many diverse organisms since its discovery in the 1920s; however, the majority of these studies were primarily based on population-wide methods. Recent advances in biological imaging, including the use of fluorescent proteins, have revolutionized the field of microbiology, and such imaging techniques allow for the study of important phenomena at the single-cell level. Here, we describe the visualization of the DNA uptake process in naturally competent Vibrio cholerae cells. More precisely, the protocol below provides instructions for the genetic engineering of V. cholerae to generate strains carrying translational fusion constructs between important competence/recombination and fluorescent proteins. Moreover, detailed steps for live cell time-lapse microscopy imaging of these strains under competence-inducing conditions are discussed for direct or indirect visualization of the DNA uptake process.