Bacterial Cell Biology, Swammerdam Institute for Life Sciences, University of Amsterdam, 1098 XH, Amsterdam, The Netherlands.
Department of Medical Microbiology and Infection Control, Amsterdam University Medical Centers - Location VUMC, 1081 HZ, Amsterdam, The Netherlands.
Commun Biol. 2021 Mar 8;4(1):306. doi: 10.1038/s42003-021-01809-8.
Transmission electron microscopy of cell sample sections is a popular technique in microbiology. Currently, ultrathin sectioning is done on resin-embedded cell pellets, which consumes milli- to deciliters of culture and results in sections of randomly orientated cells. This is problematic for rod-shaped bacteria and often precludes large-scale quantification of morphological phenotypes due to the lack of sufficient numbers of longitudinally cut cells. Here we report a flat embedding method that enables observation of thousands of longitudinally cut cells per single section and only requires microliter culture volumes. We successfully applied this technique to Bacillus subtilis, Escherichia coli, Mycobacterium bovis, and Acholeplasma laidlawii. To assess the potential of the technique to quantify morphological phenotypes, we monitored antibiotic-induced changes in B. subtilis cells. Surprisingly, we found that the ribosome inhibitor tetracycline causes membrane deformations. Further investigations showed that tetracycline disturbs membrane organization and localization of the peripheral membrane proteins MinD, MinC, and MreB. These observations are not the result of ribosome inhibition but constitute a secondary antibacterial activity of tetracycline that so far has defied discovery.
细胞样本切片的透射电子显微镜观察是微生物学中一种常用的技术。目前，在树脂包埋的细胞沉淀上进行超薄切片，这需要使用毫升到十毫升的培养物，并且得到的是随机取向的细胞切片。对于杆状细菌来说，这是有问题的，并且由于缺乏足够数量的纵向切割细胞，通常会排除对形态表型的大规模定量分析。在这里，我们报告了一种平展包埋方法，该方法能够在单个切片中观察数千个纵向切割的细胞，并且仅需要微升级的培养物体积。我们成功地将该技术应用于枯草芽孢杆菌、大肠杆菌、牛分枝杆菌和缠卷支原体。为了评估该技术定量形态表型的潜力，我们监测了抗生素诱导的枯草芽孢杆菌细胞变化。令人惊讶的是，我们发现核糖体抑制剂四环素会引起细胞膜变形。进一步的研究表明，四环素会干扰膜组织和外周膜蛋白 MinD、MinC 和 MreB 的定位。这些观察结果不是核糖体抑制的结果，而是四环素的一种次级抗菌活性，迄今为止尚未被发现。
