Partial inactivation of the mosquitocidal activity of Clostridium bifermentans serovar malaysia by extracellular proteinases

Summary

Clostridium bifermentans serovar malaysia is toxic to mosquito larvae. During large-scale preparation in a fermentor, the bacteria enter the sporulation stage after 5 h culture, whereupon high larvicidal activity is obtained (LC₅₀ 48 h on Anopheles stephensi = 3.1 × 10⁻⁵). The toxicity becomes maximal around 3–5 h later (LC₅₀ 48 h = 1.3 × 10⁻⁵) and remains unchanged until sporangium lysis. An important loss of toxicity is then observed when the cells lyse. This loss appears to be due to the fact that C. bifermentans serovar malaysia synthesizes and excretes, mainly during vegetative growth, metallo- and/or cystein-proteinases, which are active between pH 6.0 and pH 8.0. Extracellular proteinases are most likely responsible in large part for the decrease in toxic activity concomitant with cell lysis. Lysis is however prevented by addition of 10 mM ethylenediaminetetraacetic acid to the culture medium before forespore formation, and under these conditions the larvicidal activity can be maximized.