A Reproducible Plasmid Platform for Sporomusa sphaeroides to Support Bioelectrochemical Studies

Robust genetic tools are a prerequisite for causal, perturbation-based tests of redox physiology in acetogens. Here we establish practical genetic entry points for Sporomusa sphaeroides DSM 2875 under strictly anaerobic handling. We first attempted genome editing via double-crossover allelic exchange targeting pyrF using a non-replicative pUC19-based knockout construct and 5-fluoroorotic acid counterselection. Diagnostic PCR identified ΔpyrF candidates with the expected size shifts, demonstrating that homologous recombination is technically feasible in DSM 2875; however, the ΔpyrF genotype exhibited severe growth defects and could not be stably maintained over repeated passages, indicating a key limitation of a pyrF-based workflow under our current conditions. We then evaluated multiple E. coli–anaerobe shuttle plasmids for introduction and maintenance. Among the tested vectors, pJIR751 reproducibly yielded erythromycin-resistant transformants after prolonged incubation and supported serial passaging on selective media. Plasmid retention was confirmed by diagnostic PCR from liquid cultures in all tested isolates. Importantly, this maintainable plasmid platform enables genetically grounded perturbation-and-rescue experiments under electrode- or Fe0-assisted conditions, allowing mechanistic hypotheses in bioelectrochemical acetogenesis to be tested causally rather than inferred from phenotypes alone. Together, these results define current practical boundaries for S. sphaeroides genetics and establish pJIR751 as a practical foundation for downstream genetic manipulation in bioelectrochemical studies.