Unlocking Microbial Dark Matter: A Metagenome Engineering Approach

Microbiologists have only obtained <1% of Bacteria (and Archaea) species in pure cultures while the remaining species have aptly been coined as the ‘Microbial Dark Matter’(MDM). Evidently, accessing MDM culturability will pave the way for new avenues in microbial ecology and biotechnology, and we believe one reason to this unculturability is harmful generation of reactive oxygen species (ROS) at laboratory conditions. Many environmental communities experience low fluxes of
electron donors, thus generating low amounts of ROS. By contrast, laboratory conditions present a high flux of electron donors, leading to high generation of ROS, while simultaneously isolating members from one another. We hypothesize that some members of environmental communities lack some (any or all) ROS defenses. Thus, any attempt to culture said members fails at standard conditions as ROS accumulates and hampers growth. The aim of the Remicult project is to develop
a biological, gene delivery system for metagenome engineering of environmental communities. We intend to remediate culturability of members that lack ROS defenses. This delivery system employs vector constructs that harbor genes for ROS-scavenging (H₂O₂) and a fluorescent marker for cell sorting. We have a now constructed broad-host vectors harboring an operon insert, consisting of a fluorescent marker gene fused with an E. coli katG gene. Furthermore, we have established an
E. coli model for H₂O₂ MIC in ΔkatG/ΔkatEG (catalase) mutants and observed recovered growth along fluorescent signal in complementation experiments of mutants harboring the vectors. The Remicult delivery system has now been applied to soil bacteria to evaluate any remediating effect of culturability of the proposed (unculturable) species. Here, preliminary growth results show that sorted soil transconjugants can withstand the same H₂O₂ concentrations as used in the model, whereas soil isolates harboring control vector are highly challenged. Thus, generated H₂O₂ may play important role for culturability of wild type bacteria under laboratory conditions. Here, ongoing growth experiments with soil isolates harboring either vector, inoculated in standard media (without H₂O₂ addition) will reveal the true potential of this metagenome engineering concept.