Abstract

Pumping toxic substances through a cytoplasmic membrane by protein transporters known as efflux pumps represents one bacterial mechanism involved in the stress response to the presence of toxic compounds. The active efflux might also take part in exporting low-molecular-weight alcohols produced by intrinsic cell metabolism; in the case of solventogenic clostridia, predominantly acetone, butanol and ethanol (ABE). However, little is known about this active efflux, even though some evidence exists that membrane pumps might be involved in solvent tolerance. In this study, we investigated changes in overall active efflux during ABE fermentation, employing a flow cytometric protocol adjusted for Clostridia and using ethidium bromide (EB) as a fluorescence marker for quantification of direct efflux. A fluctuation in efflux during the course of standard ABE fermentation was observed, with a maximum reached during late acidogenesis, a high efflux rate during early and mid-solventogenesis and an apparent decrease in EB efflux rate in late solventogenesis. The fluctuation in efflux activity was in accordance with transcriptomic data obtained for various membrane exporters in a former study. Surprisingly, under altered cultivation conditions, when solvent production was attenuated, and extended acidogenesis was promoted, stable low efflux activity was reached after an initial peak that appeared in the stage comparable to standard ABE fermentation. This study confirmed that efflux pump activity is not constant during ABE fermentation and suggests that undisturbed solvent production might be a trigger for activation of pumps involved in solvent efflux.

Key points

• Flow cytometric assay for efflux quantification in Clostridia was established.

• Efflux rate peaked in late acidogenesis and in early solventogenesis.

• Impaired solventogenesis led to an overall decrease in efflux.

中文翻译：

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整个ABE发酵过程中拜氏梭菌外排泵活性的变化

摘要

通过被称为外排泵的蛋白质转运蛋白通过细胞质膜泵送有毒物质代表了一种细菌机制，参与了对有毒化合物的胁迫反应。主动外排还可能参与由内在细胞代谢产生的低分子量醇的输出。对于溶剂型梭状芽胞杆菌，主要是丙酮，丁醇和乙醇（ABE）。但是，尽管有证据表明膜泵可能与溶剂耐受性有关，但对这种主动外排知之甚少。在这项研究中，我们调查了ABE发酵过程中总体活性流出的变化，采用针对梭菌进行了调整的流式细胞仪协议，并使用溴化乙锭（EB）作为定量直接流出的荧光标记。观察到在标准ABE发酵过程中流出量的波动，在晚期酸生成过程中达到最大值，在早期和中期溶剂生成过程中达到高流出率，并且在晚期溶剂生成过程中EB流出率明显降低。外排活性的波动与先前研究中从各种膜出口者获得的转录组数据一致。出人意料的是，在改变的培养条件下，当减少溶剂产生并促进延长的酸生成时，在与标准ABE发酵相当的阶段出现的初始峰值之后，达到稳定的低外排活性。

关键点

•建立了用于梭状芽胞杆菌外流定量的流式细胞术测定。

•外排率在晚期酸生成和早期溶剂生成中达到峰值。

•溶剂生成受损导致外排总体减少。