Lithium Ion-dependent Energy Conservation System in Rhodobacter nitrogen fixation (Rnf) Complex-possessing Acetogen, Eubacterium callanderi KIST612

Abstract

Acetogens are anaerobic bacteria that convert one-carbon substrates such as carbon dioxide (CO₂) or carbon monoxide (CO) into acetate through the Wood–Ljungdahl pathway. These organisms rely on chemiosmotic energy conservation rather than substrate-level phosphorylation to produce ATP. The Na⁺- translocating Rhodobacter nitrogen fixation (Rnf) complex plays a central role in this process by coupling electron transfer with the translocation of Na⁺ across the membrane to generate an electrochemical gradient, which drives ATP synthesis via ATP synthase. Although Na⁺-translocating Rnf complexes are typically considered to utilize Na⁺, Li⁺ has been reported as a substitutable ion in certain Na⁺ transport proteins, including the homologous Na⁺-NQR complex. In Acetobacterium woodii DSM 1030T, Li⁺ utilization via the Rnf complex has been experimentally confirmed, however, whether this is strain-specific or conserved remains unclear. In this study, Eubacterium callanderi KIST612 was selected as a model strain because it is a representative acetogen with clearly characterized Na⁺-translocating Rnf complex. Proteomic analysis revealed significant upregulation of the Rnf complex and ATP synthase under autotrophic conditions, indicating potential activation of the chemiosmotic energy conservation system. Based on these data, acetate production was evaluated under H₂/CO₂ conditions supplemented with Na⁺, Li⁺, or K⁺. The results showed that acetate production was enhanced under both Na⁺ and Li⁺ conditions compared to the control, suggesting that Li⁺ could be utilized for ion gradient necessary for ATP synthesis and carbon fixation via the Wood–Ljungdahl pathway. Growth profiling further supported this observation, with robust growth under Na⁺ and Li⁺ conditions and no growth under K⁺ or control conditions, confirming functional ATP generation with Li⁺. Additionally, structural analysis of six representative Na⁺-translocating Rnf complex-possessing acetogens, including A. woodii DSM 1030T and E. callanderi KIST612, revealed conserved Na⁺-binding residues (T111RnfA and V106RnfE) and four cysteine ligands coordinating the [2Fe–2S] cluster essential for ion translocation. These features were preserved not only in the Li⁺-utilizing strains but also in other examined acetogens, suggesting that the ability to utilize Li⁺ could be a conserved characteristic of Na⁺-translocating Rnf complexes. This study expands our understanding of ion selectivity in Na⁺-translocating Rnf complexes and experimentally demonstrates that acetogenic energy conservation systems could utilize Li⁺. These findings provide foundational insights for the future design of bioenergy production systems and emerging ion-based bioelectrochemical energy storage technologies.