A flat embedding method for transmission electron microscopy reveals an unknown mechanism of tetracycline

Michaela Wenzel,Jan R T Van Weering,Maroeska J Burggraaf,Wilbert Bitter,Biwen Wang,Marien P Dekker,Leendert W Hamoen

doi:10.1038/s42003-021-01809-8

Abstract

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.

Highlights

Transmission electron microscopy of cell sample sections is a popular technique in microbiology
Light microscopy studies of bacteria commonly use thin agarose layers to immobilize cells[11,12]. These cells are well-aligned in a single plane, allowing large-scale quantification of phenotypic changes. We wondered whether this immobilization technique could be adapted for Transmission electron microscopy (TEM) embedding, which would solve the issue of randomly sectioned bacteria and at the same time drastically reduce the required sample volume
When we examined TEM pictures of bacteria prepared with the classical pellet embedding method, we found on average only six fully longitudinally sectioned bacteria per image (Fig. 1b)

Summary

Introduction

Transmission electron microscopy of cell sample sections is a popular technique in microbiology. This is a critical limitation when examining rod-shaped and other non-coccoid bacterial species, since the vast majority of cells are randomly cross-sectioned, and the number of complete longitudinally cut cells is generally so low that robust quantification and population-wide studies are not feasible Another limitation is that acquiring a concentrated cell pellet often requires relatively large culture volumes typically in the range of 10 to 50 ml[6,8,9]. We have successfully used this method with the Gram-positive bacterium Bacillus subtilis, the Gram-negative bacterium Escherichia coli, the tuberculosis vaccine strain Mycobacterium bovis Bacillus Calmette–Guérin (BCG), and the cell wall-less mycoplasma species Acholeplasma laidlawii This flat embedding technique allowed the quantification of morphological changes in bacteria treated with different antibiotics. This additional mechanism of action has remained hidden for over 50 years despite the fact that tetracyclines are one of the most commonly used antibiotic groups in both human and veterinary medicine[10]

Methods

Results

Conclusion