Development of an efficient recombineering system for Aeromonas

The proteins Redαβγ from the lambda phage and RecET enzymes from the Rac prophage are highly effective for accurate and efficient genetic engineering, but their application has been restricted to a few prokaryotic organisms due to host specificities. Species of the genus Aeromonas are commonly found as integral parts of indigenous microbial communities in water bodies. This group of bacteria is of interest for both its opportunistic pathogenicity in humans and animals and its capability to generate industrial enzymes, biodegradable plastics, and environmental remediation strategies. Currently, gene function analysis in Aeromonas species relies primarily on suicide vector-mediated homologous recombination and random transposon mutagenesis. However, these methods are either too laborious or lack the precision required for efficient functional genomics. To address this limitation, we developed a host-specific recombineering system by harnessing redABSAh, a redαβ-like operon identified in a multidrug-resistant Aeromonas hydrophila strain. An efficient and streamlined workflow was established in A. bivalvium, with 200-bp homology arms and 300 ng of DNA determined to yield high recombinant frequencies. The system’s utility for functional genomics was demonstrated through a case study dissecting flagellar motility, which revealed differential roles in motility for flhAAb and lafA1A2Ab genes in A. bivalvium. Furthermore, we validated the system’s broad utility by achieving efficient marker-free editing and demonstrating its reliable cross-species application in A. veronii and A. schubertii. Our findings establish the first genus-specific recombinase platform for Aeromonas, overcoming previous molecular manipulation constraints. The system’s broad compatibility across strains significantly enhances functional genomics research capabilities, enabling efficient and precise genetic investigations in this ecologically and medically important bacterial group. This breakthrough expands the toolkit for both fundamental research and biotechnological applications.