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

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.


Supplementary Information
A flat embedding method for transmission electron microscopy reveals an unknown mechanism of tetracycline Michaela Wenzel, Marien P. Dekker, Biwen Wang, Maroeska J. Burggraaf, Wilbert Bitter, Jan R. T. van 11 . Accordingly, ampicillin-treated cells displayed partly disintegrated cell walls. Daptomycin was recently shown to hamper cell wall synthesis by targeting membrane microdomains that harbor the cell wall synthetic machinery, causing them to accumulate into lipid II-enriched foci 12,13 . In line, daptomycin-treated cells showed aberrant local cell wall thickening. The antimicrobial peptide MP196 caused intracellular cell wall structures and membrane vesicles, reflecting its dual mechanism of targeting membrane function and cell wall synthesis 14 . Nitrofurantoin is thought to kill cells by an unspecific mechanism involving oxidative damage 15 . Cells treated with this antibiotic lacked a nucleoid and showed membrane aberrations, which is consistent with oxidative damage to these cellular structures. Tetracycline inhibits the bacterial ribosome 16 . Surprisingly, we consistently observed membrane lesions in tetracycline-treated cells. Anhydrotetracycline, an analogue of tetracycline, which is thought to rather target the cell membrane than the ribosome 17 , caused similar lesions.

Scale bars 1 µm (A) and 250 µm (B).
Supplementary Supplementary Figure 15: Inhibition of translation does not cause delocalization of the membrane potential-dependent membrane proteins MinD and MinC. B. subtilis LB318, expressing GFP-MinD and mCherry-MinC, was treated with 20 µg/ml chloramphenicol, 10 µg/ml kanamycin, or 1 µg/ml gramicidin for 20 min prior to microscopy. Note that LB318 carries both a chloramphenicol and kanamycin resistance cassette. Therefore, twice the concentrations used for antibiotic selection were chosen for microscopy. Scale bar 2 µm.
Supplementary Figure 16: Inhibition of translation does not cause delocalization of MreB. B. subtilis TNVS205, expressing mCherry-MreB, was treated with 20 µg/ml chloramphenicol, 3 µg/ml kanamycin, or 1 µg/ml gramicidin for 20 min prior to microscopy. Note that TNVS205 carries a chloramphenicol resistance cassette. Therefore, twice the concentration used for antibiotic selection was chosen for microscopy. Scale bar 2 µm.
Supplementary Figure 17: Inhibition of translation does not diminish fluid membrane domains. B. subtilis 168 was treated with 15 µg/ml chloramphenicol or 3 µg/ml kanamycin 30 min prior to microscopy. Small effects are expected since RIFs depend on the growth phase 20 and a reduced growth rate caused by antibiotic treatment is likely to have secondary effects on RIFs. In line, RIFs were less clear after 30 min treatment with chloramphenicol and kanamycin compared to the untreated control. However, clustering or diminishing of RIFs was not observed. Scale bar 2 µm.
Supplementary Figure 18: Effects of different tetracycline concentrations on B. subtilis 168 and PG112. The tet-4 mutant strain PG112 shows the exact same phenotype as the 168 wild type at all concentrations, further corroborating the notion that the membrane activity of tetracycline is independent of ribosome inhibition. Scale bar 2 µm.