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

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.

Isolation of Persister Cells of Bacillus subtilis and Determination of Their Susceptibility to Antimicrobial Peptides

“…Upregulated liaI indicated that increased cell envelop stress was associated to B. subtilis persister formation and/or the maintenance of this persistent physiological state. This result is consistent with earlier studies showing that cell envelope stress was strongly induced by the cell wall targeting antibiotics vancomycin [49]; the membrane-active chemical CCCP [50] and tetracycline [34] also led to increased cell envelope stress response. In response to membrane-damaging agents, LiaH (encoded by liaI) contributes to the maintenance of membrane integrity and prevents depolarization in B. subtilis [51].…”

“…Bacillus subtilis strain PS832 was treated with 100-fold the minimum inhibitory concentration (MIC) of four antimicrobial compounds with different killing mechanisms: 12.5 μg/mL vancomycin (inhibiting the lipid II cycle of cell wall biosynthesis [ 30 ]); 6.25 μg/mL enrofloxacin (leading to double-stranded DNA breaks [ 31 ]); 100 μg/mL Carbonyl cyanide m -chlorophenyl hydrazone (CCCP, a proton-ionophore that causes the dissipation of the proton motive force and then inhibits the activity of bacterial ATPases [ 32 ]); and 400 μg/mL tetracycline (reversibly inhibiting protein synthesis [ 33 ] and disturbing membrane organization [ 34 ]). Time-kill assays showed a biphasic killing curve ( Figure 1 A), indicating that while most of the vegetative cells were killed by the tested antimicrobial compounds, a subpopulation survived, which matched the definition of persisters [ 35 ].…”

Imaging Giant Vesicle Membrane Domains with a Luminescent Europium Tetracycline Complex

“…For example, tetracycline and derivatives in the presence of calcium have been shown to induce membrane invaginations, disrupt the spatial distribution of peripheral membrane proteins, and alter the size and distribution of regions of increased fluidity (RIFs) in gram-positive bacteria. [31] Furthermore, under physiological conditions free tetracycline concentrations are negligible, with Mg 2+ and Ca 2+ tetracycline complexes being the most prevalent forms. [32,33] Metal complexation of tetracycline decreases its bioavailability through altered interactions with gastrointestinal tract tissue.…”

Cytoplasmic membrane thinning observed by interfacial dyes is likely a common effect of bactericidal antibiotics

“…This delayed, direct membrane effect of tetracycline was unexpected (28). However, tetracycline affecting membranes directly in a mechanism independent of ribosome inhibition has been recently characterised (29).…”