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The use of CRISPR/Cas (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR associated protein) for sequence-specific elimination of bacteria or resistance genes is a powerful tool for combating antibiotic resistance. However, this approach requires efficient delivery of CRISPR/Cas DNA cassette(s) into the targeted bacterial population. Compared to phage transduction, plasmid conjugation can deliver DNA to a broader host range but often suffers from low delivery efficiency. Here, we developed multi-plasmid conjugation systems for efficient CRISPR/Cas delivery, target DNA elimination and plasmid replacement. The CRISPR/Cas system, delivered via a broad-host-range R1162 mobilizable plasmid, specifically eliminated the targeted plasmid in recipient cells. A self-transmissible RK2 helper plasmid facilitated the spread of mobilizable CRISPR/Cas. The replacement of the target plasmid with another plasmid from the same compatibility group helped speed up target plasmid elimination especially when the target plasmid was also mobilizable. Together, we showed that up to 100% of target plasmid from the entire recipient population could be replaced even at a low (1:180) donor-to-recipient ratio and in the absence of transconjugant selection. Such an ability to modify genetic content of microbiota efficiently in the absence of selection will be critical for future development of CRISPR antimicrobials as well as genetic tools for in situ microbiome engineering.

BACKGROUND Nitrate‐contaminated water is a concerning environmental and health problem in agricultural areas. Due to its low organic carbon content, such water cannot be purified using a conventional bioreactor with heterotrophic denitrification. Low‐cost bio‐electrochemical reactors were established with the aim of enhancing denitrification performance by cooperation of heterotrophic and hydrogenotrophic denitrification. RESULTS At the lowest C/N ratio of 1.0, the bioreactor reached only 67.4% for NO3‐N removal and 47.5% for total N removal (total N removal rate of ca 1.2 mg L−1 h−1), whereas the Cu reactor (bio‐electrochemical reactor using copper wire as cathode) achieved the best efficiencies of 73.7% and 53.8% for NO3‐N and total N removal (total N removal rate of ca 1.3 mg L−1 h−1), and followed by the SS reactor (bio‐electrochemical reactor using stainless steel wire as cathode). On the other hand, the greatest total organic carbon (TOC) removal efficiency was observed for the bioreactor and followed by the SS reactor and the Cu reactor. The low TOC consumption of 1.2–1.4 mg‐TOC (mg‐N)−1 in the Cu reactor and the SS reactor indicated the enhanced denitrification from coexisting hydrogenotrophic denitrification. In addition, the performance of hydrogenotrophic denitrification was improved by increasing the applied current from 10 to 30 mA. CONCLUSIONS The abundance of autotrophic denitrifying bacteria (identified as hydrogenotrophs) was higher than that of heterotrophic denitrifying bacteria in both the Cu reactor and the SS reactor. Archromobacter and Flavobacterium were the most dominant genera in the biofilm of the Cu reactor cathode and in the suspended sludge, respectively. Hydrogenophaga were detected in both biomass samples, but were absent from the biomass samples of the conventional bioreactor. © 2021 Society of Chemical Industry (SCI).

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