Investigation of the Effect of Chlorine Dioxide on Spores of the Mosquito-killing Bacterium Lysinibacillus sphaericus by Single-cell Analysis

doi:10.16409/j.cnki.2095-039x.2021.09.028

Abstract

Abstract: Lysinibacillus sphaericus (Ls) is a widely used biological control agent that produces insecticidal proteins with high activity against mosquito larvae. In practical applications, Ls may encounter unfavorable environmental factors or different disinfectants; chlorine dioxide (ClO₂) is an efficient disinfectant. In this study, the individual chlorine dioxide-treated Ls spores were observed by single-cell Raman spectroscopy and the germination dynamics of spores was monitored by differential interference contrast microscopy to investigate the effects of chlorine dioxide on the structure and composition of Ls spores and proteins involved in spore germination, as well as the spore-killing mechanism of chlorine dioxide. Thirty six to eighty six percent of the spores were nonculturable but remained their CaDPA at 5 min post treatment with 0.05%-0.3% chlorine dioxide. The intensity of peak derives from pyridine-2, 6-dicarboxylic acid, DPA (1017 cm^-1) decreased with increasing concentration of the treatment solution, while the intensity of peak from α-helix of protein (1652 cm^-1) did not change significantly. Treatment remarkably altered the germination dynamics of individual spores triggered by L-alanine or dodecylamine, significantly prolonging the time to initiate rapid release of CaDPA, to complete CaDPA release, and to complete hydrolysis of spore cortex. In contrast, exogenous CaDPA was only able to trigger the germination of spores treated with low concentrations of chlorine dioxide (0.05%) and was significantly delayed. These results show that chlorine dioxide does not destroy the inner membrane of spores directly, but severely impairs the function of germination-related proteins, especially the cortex-lytic enzymes. This may be the reason that the chlorine dioxide- killed spores can undergo the initial steps of germination but cannot go further to grow into vegetative cells.

Key words: Lysinibacillus sphaericus, chlorine dioxide, Raman spectrum, differential interference contrast imaging, spore germination, single cell analysis

CLC Number:

TQ453.5

LI Cuimei, WANG Xiaochun, ZhANG Xiujuan, LIU Junxian, WANG Guiwen. Investigation of the Effect of Chlorine Dioxide on Spores of the Mosquito-killing Bacterium Lysinibacillus sphaericus by Single-cell Analysis[J]. Chinese Journal of Biological Control, 2022, 38(3): 565-572.

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