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Prokaryotic evolution is driven by random mutations and horizontal gene transfer (HGT). HGT occurs via transformation, transduction, or conjugation. We have previously shown that in syntrophic cocultures of Clostridium acetobutylicum and Clostridium ljungdahlii, heterologous cell fusion leads to a large-scale exchange of proteins and RNA between the two organisms. Here, we present evidence that heterologous cell fusion facilitates the exchange of DNA between the two organisms. Using selective subculturing, we isolated C. acetobutylicum cells which acquired and integrated into their genome portions of plasmid DNA from a plasmid-carrying C. ljungdahlii strain. Limiting-dilution plating and DNA methylation data based on PacBio Single-Molecule Real Time (SMRT) sequencing support the existence of hybrid C. acetobutylicum/C. ljungdahlii cells. These findings expand our understanding of multi-species …