Multi-omics analysis reveals the genetic basis for rapid CO₂ utilization in the acetogenic bacterium Sporomusa...

Sporomusa sphaeroides KIAC is a novel acetogen isolated from cattle feces that exhibits rapid CO₂ utilization. To investigate the molecular basis of this phenotype, we performed a comprehensive multi-omics analysis, including Genome-seq, RNA-seq, dRNA-seq, and Term-seq, to map its transcriptome architecture. We identified 2,158 transcription start sites and 2,275 transcript 3' ends, enabling high-resolution reconstruction of the transcriptional landscape and associated regulatory features. This analysis uncovered key cis-regulatory elements and an expanded regulatory role for the alternative sigma factor SigH in controlling acetogenesis-related genes. Notably, KIAC harbors nine functionally diverse hydrogenases-a greater diversity than observed in other acetogens-likely contributing to its rapid CO₂ utilization. Heterologous expression of KIAC-derived hydrogenases in Eubacterium limosum led to doubled H₂ and CO₂ consumption rates, increased growth rates, and notably, the first reported butyrate production under energy-limited H₂/CO₂ conditions. These improvements stem from enhanced H₂ oxidation, which supplies additional reducing equivalents for growth and biochemical production. Our findings provide critical insights into the genetic basis for rapid autotrophic growth in acetogens. The discovery of the expanded regulatory role of SigH and the energetic advantages of diverse hydrogenases offers new strategies for enhanced CO₂ bioconversion of acetogens.IMPORTANCEAcetogens offer a promising solution for sustainable CO₂ bioconversion into multicarbon biochemicals through the Wood-Ljungdahl pathway, the most energy-efficient carbon fixation route known in nature. However, an incomplete understanding of their metabolism and regulatory systems has limited metabolic engineering efforts to achieve superior CO₂ fixation efficiency. In this study, we investigated Sporomusa sphaeroides KIAC, a newly isolated acetogen with rapid CO₂ utilization, to uncover the molecular mechanisms underlying its superior performance. By revealing an expanded regulatory role for an alternative sigma factor and a highly diverse set of hydrogenases, our findings provide a foundation for engineering acetogens with enhanced CO₂ conversion efficiency under energy-limited conditions.