Fluorescence Recovery Allows the Implementation of a Fluorescence Reporter Gene Platform Applicable for the Detection and Quantification of Horizontal Gene Transfer in Anoxic Environments.

The study of horizontal gene transfer (HGT) in microbial communities has been revolutionized by significant advances in cultivation-independent methods based on fluorescence reporter gene technologies. Recently, the combination of these novel approaches with flow cytometry has presented itself as one of the most powerful tools to study the spread of mobile genetic elements (MGEs) in the environment. However, the use of fluorescent markers, like green fluorescent protein (GFP) and mCherry, is limited by environmental constraints, such as oxygen availability and pH levels, that affect the correct maturation of their fluorophores. Few studies have characterized the effects of such environmental conditions in a systematic way, and the sheer amount of distinct protein variants requires each system to be examined in an individual fashion. The lack of efficient and reliable markers to monitor HGT in anaerobic environments, coupled to the abundance of ecologically and clinically relevant oxygen-deprived niches in which bacteria thrive, calls for the urgent development of suitable tools that permit its study. In an attempt to devise a process that allows the implementation of the mentioned dual-labeling system to anoxic milieus, the aerobic fluorescence recovery of mCherry and GFPmut3, as well as the effect of pH on their fluorescence intensities, was studied. The findings present a solution to an intrinsic problem that has long hampered the utilization of this system, highlight its pH limitations, and provide experimental tools that will help broaden its horizon of application to other fields.