Comparative Analysis of Toxigenic and Megaplasmid-Free Clostridium tetani Strains Reveals Physiological Transitions Linked to Tetanus Toxin Production

Understanding Clostridium tetani metabolism and physiology is essential for identifying factors controlling toxin synthesis. We compared a toxigenic C. tetani strain with a derivative strain lacking the toxin-encoding megaplasmid carrying tetR and tetX genes involved in toxin regulation and production. Both strains were cultured under identical strict anaerobic conditions in complex media. The toxigenic strain exhibited four successive fermentation phases, including rapid growth, toxin accumulation-associated slower growth, cell lysis, and toxin maturation. Cultivation yielded 0.45 g/L of biomass, for a final toxin titer of 45 Lf/mL. The megaplasmid-free strain demonstrated no toxin production or cell lysis, yielding a higher biomass concentration (0.75 g/L) maintained for 150 h. Flow cytometry was applied for the first time to monitor C. tetani population dynamics throughout the culture. The toxigenic strain exhibited notable morphological heterogeneity and transient filamentous cells during rapid growth, followed by two smaller-cell subpopulations emerging during toxin production. The megaplasmid-free strain rapidly stabilized into a single homogeneous population. Membrane integrity remained largely preserved, while extracellular toxin became detectable, suggesting that extracellular toxin detection before cell lysis onset may involve an active export mechanism. Overall, these findings reveal the physiological transitions associated with megaplasmid presence and provide new insights into the cellular processes underlying tetanus toxin production.