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.IMPORTANCE Many anaerobic environments, like the gastrointestinal tract, anaerobic digesters, and the interiors of dense biofilms, have been shown to be hotspots for horizontal gene transfer (HGT). Despite the increasing wealth of reports warning about the alarming spread of antibiotic resistance determinants, to date, HGT studies mainly rely on cultivation-based methods. Unfortunately, the relevance of these studies is often questionable, as only a minor fraction of bacteria can be cultivated. A recently developed approach to monitoring the fate of plasmids in microbial communities is based on a fluorescence dual-labeling system and allows the bypassing of cultivation. However, the fluorescent proteins on which it is founded are constrained by pH levels and by their strict dependence on oxygen for the maturation of their fluorophores. This study focused on the development and validation of an appropriate aerobic fluorescence recovery (AFR) method for this platform, as this embodies the missing technical link impeding its implementation in anoxic environments.

Conjugative plasmids inhibit extracellular electron transfer in Geobacter sulfurreducens.

Geobacter sulfurreducens is part of a specialized group of microbes with the unique ability to exchange electrons with insoluble materials, such as iron oxides and electrodes. Therefore, G. sulfurreducens plays an essential role in the biogeochemical iron cycle and microbial electrochemical systems. In G. sulfurreducens this ability is primarily dependent on electrically conductive nanowires that link internal electron flow from metabolism to solid electron acceptors in the extracellular environment. Here we show that when carrying conjugative plasmids, which are self-transmissible plasmids that are ubiquitous in environmental bacteria, G. sulfurreducens reduces insoluble iron oxides at much slower rates. This was the case for all three conjugative plasmids tested (pKJK5, RP4 and pB10). Growth with electron acceptors that do not require expression of nanowires was, on the other hand, unaffected. Furthermore, iron oxide reduction was also inhibited in Geobacter chapellei, but not in Shewanella oneidensis where electron export is nanowire-independent. As determined by transcriptomics, presence of pKJK5 reduces transcription of several genes that have been shown to be implicated in extracellular electron transfer in G. sulfurreducens, including pilA and omcE. These results suggest that conjugative plasmids can in fact be very disadvantageous for the bacterial host by imposing specific phenotypic changes, and that these plasmids may contribute to shaping the microbial composition in electrode-respiring biofilms in microbial electrochemical reactors.