Department of Plant Sciences, School of Life Sciences, University of Hyderabad, P.O. Central University, Hyderabad 500046, India.
Bacterial Discovery Laboratory, Centre for Environment, Institute of Science and Technology, J.N.T. University Hyderabad, Hyderabad 500085, India.
J Appl Microbiol. 2023 Nov 1;134(11). doi: 10.1093/jambio/lxad238.
The purpose of the present study was to understand the possible events involved in the toxicity of hydrogen peroxide (H2O2) to wild and sporulene-deficient spores of Bacillus subtilis, as H2O2 was previously shown to have deleterious effects.
The investigation utilized two strains of B. subtilis, namely the wild-type PY79 (WT) and the sporulene-deficient TB10 (ΔsqhC mutant). Following treatment with 0.05% H2O2 (v/v), spore viability was assessed using a plate count assay, which revealed a significant decrease in cultivability of 80% for the ΔsqhC mutant spores. Possible reasons for the loss of spore viability were investigated with microscopic analysis, dipicholinic acid (DPA) quantification and propidium iodide (PI) staining. Microscopic examinations revealed the presence of withered and deflated morphologies in spores of ΔsqhC mutants treated with H2O2, indicating a compromised membrane permeability. This was further substantiated by the absence of DPA and a high frequency (50%-75%) of PI infiltration. The results of fatty acid methyl ester analysis and protein profiling indicated that the potentiation of H2O2-induced cellular responses was manifested in the form of altered spore composition in ΔsqhC B. subtilis. The slowed growth rates of the ΔsqhC mutant and the heightened sporulene biosynthesis pathways in the WT strain, both upon exposure to H2O2, suggested a protective function for sporulenes in vegetative cells.
Sporulenes serve as a protective layer for the inner membrane of spores, thus assuming a significant role in mitigating the adverse effects of H2O2 in WT B. subtilis. The toxic effects of H2O2 were even more pronounced in the spores of the ΔsqhC mutant, which lacks this protective barrier of sporulenes.
本研究旨在了解过氧化氢（H2O2）对野生型和芽孢缺陷型枯草芽孢杆菌孢子毒性的可能作用机制，因为之前的研究表明 H2O2 具有有害作用。
本研究利用了两种枯草芽孢杆菌菌株，即野生型 PY79（WT）和芽孢缺陷型 TB10（ΔsqhC 突变体）。用 0.05% H2O2（v/v）处理后，用平板计数法评估孢子活力，结果显示 ΔsqhC 突变体孢子的可培养性显著下降了 80%。用显微镜分析、二吡啶羧酸（DPA）定量和碘化丙啶（PI）染色来研究孢子活力丧失的可能原因。显微镜检查显示，用 H2O2 处理的 ΔsqhC 突变体孢子存在干瘪和塌陷的形态，表明细胞膜通透性受损。这进一步得到了 DPA 缺失和 PI 渗透高频率（50%-75%）的证实。脂肪酸甲酯分析和蛋白质谱分析的结果表明，在 ΔsqhC 枯草芽孢杆菌中，H2O2 诱导的细胞反应增强表现为孢子组成的改变。H2O2 暴露后，ΔsqhC 突变体的生长速度减慢，WT 菌株的芽孢烯生物合成途径增强，这表明芽孢烯在营养细胞中具有保护功能。
芽孢烯是孢子内膜的保护层，因此在 WT 枯草芽孢杆菌中减轻 H2O2 的不利影响方面发挥着重要作用。缺乏芽孢烯这种保护屏障的 ΔsqhC 突变体孢子，H2O2 的毒性作用更为明显。
