Traditionally, microbial biotechnology has primarily relied on bacteria grown in monoculture. However, most microbes likely evolved to occupy niches within larger communities, making it unsurprising that certain metabolically complementary bacteria can “team up” to outperform individual microbes. We recently showed that coculturing Clostridium acetobutylicum (Cac) and Clostridium ljungdahlii (Clj) increases substrate carbon and electron conversion via CO2 and H2 capture and synthesizes valuable products, such as isopropanol and 2,3-butanediol, that neither species can make independently. Further study suggests that this metabolic phenotype occurs due to interspecies cell fusion events involving large scale exchange of cytoplasmic content, including RNA and protein, between Cac and Clj. Subculturing these fused coculture populations under specific selection leads to the evolution of replicating hybrid cell lines containing DNA from and exhibiting characteristics of both organisms. The mechanism by which these fusions occur, and how they contribute to the creation of persistent hybrid microbial cells, is unknown. As a first step towards exploring this mechanism, we apply RNA sequencing (RNAseq) to identify genes differentially expressed in the Cac-Clj coculture relative to the monocultures. Preliminary results show differential expression of genes involved in DNA transcription and translocation, chemotactic cell-cell sensing, cytoskeleton assembly, amino acid synthesis, cofactor uptake, ATP production, hydrogen uptake, electron transfer, and central carbon metabolism across both species. These changes are particularly pronounced in Clj, which differentially regulates &gt;20% of its genome in coculture. Combining transcriptomics with metabolite and species ratio profiling produces a more comprehensive picture of how cytoplasmic metabolite exchange influences gene expression (and vice versa) in this synthetic microbial consortium. Additional RNAseq experiments, in which monoculture controls were grown separated by a permeable membrane, identified genes specifically involved in interspecies cell contact and fusion. These results have important implications both for microbial biotechnology and fundamental studies of bacterial evolution, illustrating how the syntrophic exchange of substrates, cofactors, and metabolic intermediates can induce metabolism and gene expression to dynamically “couple,” improving chemical biosynthesis and accelerating community evolution.

Mechanistic understanding of syntrophic Clostridium coculture using transcriptomic and metabolic analyses

ACS Spring 2022

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