School of Environment and Energy, South China University of Technology, Guangzhou, People's Republic of China.
School of Biology and Biological Engineering, South China University of Technology, Guangzhou, People's Republic of China.
Appl Environ Microbiol. 2020 Jul 2;86(14). doi: 10.1128/AEM.00345-20.
Accurate determination of microbial viability can be crucial in microbe-dominated biosystems. However, the identification of metabolic decay in bacterial cells can be elaborate and difficult. We sought to identify apoptosis-like bacterial processes by using annexin V-fluorescein isothiocyanate (FITC) (AVF), a probe typically used to stain phosphatidylserine (PS) on exposed cell membranes. The bacterial cell wall provides a barrier that is responsible for low efficiency of direct PS staining of decayed bacterial cells. This can be overcome by pretreatment of the bacteria with 70% ethanol, which fixates the bacteria and preserves the PS status, combined with lysozyme treatment to hydrolyze the cell wall. That treatment improved the efficiency of AVF staining considerably, as shown for pure strains of an sp. and a sp. Using this method, decayed bacterial cells (induced by starvation) were more strongly stained, indicating externalization of PS to a greater extent than seen for cells harvested at logarithmic growth. A multispecies microbial sludge was artificially decayed by heat treatment or alternating anoxic-oxic treatment, which also induced increased AVF staining, again presumably via decay-related PS externalization. The method developed proved to be efficient for identification of bacterial decay and has potential for the evaluation of multispecies bacterial samples from sources like soil matrix, bioaerosol, and activated sludge. Since the externalization of phosphatidylserine (PS) is considered a crucial characteristic of apoptosis, we sought to identify apoptosis-like decay in bacterial cells by PS staining using AVF. We show that this is possible, provided the bacteria are pretreated with ethanol plus lysozyme to remove a physical staining barrier and preserve the original, decay-related externalization of PS. Our work suggests that PS externalization occurs in starved bacteria and this can be quantified with AVF staining, providing a measure of bacterial decay. Since PS is the common component of the lipid bilayer in bacterial cell membranes, this approach also has potential for evaluation of cell decay of other bacterial species.
准确测定微生物的生存能力在以微生物为主的生物系统中至关重要。然而，鉴定细菌细胞中的代谢衰退可能比较复杂和困难。我们试图通过使用膜联蛋白 V-异硫氰酸荧光素（AVF）鉴定类似细胞凋亡的细菌过程，AVF 是一种通常用于染色暴露细胞膜上磷脂酰丝氨酸（PS）的探针。细菌细胞壁形成了一道屏障，导致直接对衰退细菌进行 PS 染色的效率较低。这可以通过用 70%乙醇预处理细菌来克服，乙醇固定细菌并保持 PS 状态，同时用溶菌酶处理以水解细胞壁。该处理大大提高了 AVF 染色的效率，这在 sp. 和 sp. 的纯菌株中得到了证明。使用这种方法，饥饿诱导的衰退细菌细胞被强烈染色，表明 PS 外排在更大程度上，比对数生长期收获的细胞更为明显。通过热处理或交替缺氧-好氧处理人为地使多物种微生物污泥衰退，这也导致 AVF 染色增加，可能也是通过与衰退相关的 PS 外排。所开发的方法已被证明可有效鉴定细菌衰退，并有可能用于评估来自土壤基质、生物气溶胶和活性污泥等来源的多物种细菌样品。由于磷脂酰丝氨酸（PS）的外排在细胞凋亡中被认为是一个关键特征，因此我们试图通过 AVF 染色鉴定细菌细胞中的类似细胞凋亡的衰退。我们表明，这是可能的，前提是细菌先用乙醇加溶菌酶预处理以去除物理染色障碍并保持原始的、与衰退相关的 PS 外排。我们的工作表明，PS 外排在饥饿细菌中发生，这可以通过 AVF 染色来定量，提供了细菌衰退的一种测量方法。由于 PS 是细菌细胞膜脂质双层的共同组成部分，因此这种方法也有可能用于评估其他细菌物种的细胞衰退。
