Separate, separated, and together: the transcriptional program of the Clostridium acetobutylicum-Clostridium ljungdahlii syntrophy leading to interspecies cell fusion

Journal Article · Tue May 20 00:00:00 EDT 2025 · mSystems

DOI:https://doi.org/10.1128/msystems.00030-25· OSTI ID:2563431

Willis, Noah B.; Papoutsakis, Eleftherios T.; Nikel, ed., Pablo Ivan

ABSTRACT <p> Syntrophic cocultures (hitherto assumed to be commensalistic) of <italic toggle='yes'>Clostridium acetobutylicum</italic> and <italic toggle='yes'>Clostridium ljungdahlii</italic> , whereby CO ₂ and H ₂ produced by the former feed the latter, result in interspecies cell fusion involving large-scale exchange of protein, RNA, and DNA between the two organisms. Although mammalian cell fusion is mechanistically dissected, the mechanism for such microbial-cell fusions is unknown. To start exploring this mechanism, we used RNA sequencing to identify genes differentially expressed in this coculture using two types of comparisons. One type compared coculture to the two monocultures, capturing the combined impact of interactions through soluble signals in the medium and through direct cell-to-cell interactions. The second type compared membrane-separated versus -unseparated cocultures, isolating the impact of interspecies physical contact. While we could not firmly identify specific genes that might drive cell fusion, consistent with our hypothesized model for this interspecies microbial cell fusion, we observed differential regulation of genes involved in <italic toggle='yes'>C. ljungdahlii’s</italic> autotrophic Wood-Ljungdahl pathway metabolism and genes of the motility machinery. Unexpectedly, we also identified differential regulation of biosynthetic genes of several amino acids, and notably of arginine and histidine. We verified that they are produced by <italic toggle='yes'>C. acetobutylicum</italic> and are metabolized by <italic toggle='yes'>C. ljungdahlii</italic> to its growth advantage. These and other findings, and notably upregulation of <italic toggle='yes'>C. acetobutylicum</italic> ribosomal-protein genes, paint a more complex syntrophic picture and suggest a mutualistic relationship, whereby beyond CO ₂ and H ₂ , <italic toggle='yes'>C. acetobutylicum</italic> feeds <italic toggle='yes'>C. ljungdahlii</italic> with growth-boosting amino acids, while benefiting from the H ₂ utilization by <italic toggle='yes'>C. ljungdahlii</italic> . </p> <sec> <title>IMPORTANCE

The construction and study of synthetic microbial cocultures is a growing research area due to the untapped potential of defined multi-species industrial bioprocesses and the utility of defined cocultures for generating insight into complex, undefined, natural microbial consortia. Our previous work showed that coculturing C. acetobutylicum and C. ljungdahlii leads to a unique metabolic phenotype (production of isopropanol) and heterologous cell fusion events. Here, we used RNAseq to explore genes involved in and impacted by these fusions. First, we compared gene expression in coculture to each monoculture. Second, we utilized a transwell system to compare gene expression in mixed cocultures to cocultures with both species physically separated by a permeable membrane, isolating the impact of interspecies “touching” on the transcriptome. This study deepens our mechanistic understanding of the C. acetobutylicum-C. ljungdahlii coculture phenotype, laying the groundwork for reverse genetic studies of heterologous cell fusion in Clostridium cocultures.

• Sponsoring Organization: USDOE Advanced Research Projects Agency - Energy (ARPA-E)
• Grant/Contract Number: AR0001505
• OSTI ID: 2563431
• Journal Information: mSystems, Journal Name: mSystems Journal Issue: 5 Vol. 10; ISSN 2379-5077
• Publisher: American Society for Microbiology
• Country of Publication: United States
• Language: English