TY - JOUR
T1 - A flat embedding method for transmission electron microscopy reveals an unknown mechanism of tetracycline
AU - Wenzel, M.
AU - Dekker, M.P.
AU - Wang, B.
AU - Burggraaf, M.J.
AU - Bitter, W.
AU - van Weering, J.R.T.
AU - Hamoen, L.W.
N1 - With supplementary files.
PY - 2021/3/8
Y1 - 2021/3/8
N2 - 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.
AB - 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.
KW - Anti-Bacterial Agents/pharmacology
KW - Bacillus subtilis/drug effects
KW - Bacterial Proteins/metabolism
KW - Cell Membrane/drug effects
KW - Membrane Proteins/metabolism
KW - Microscopy, Electron, Transmission
KW - Microtomy
KW - Tetracycline/pharmacology
KW - Tissue Embedding
UR - https://pure.uva.nl/ws/files/69279857/42003_2021_1809_MOESM1_ESM.pdf
UR - https://pure.uva.nl/ws/files/69279859/42003_2021_1809_MOESM2_ESM.pdf
UR - https://pure.uva.nl/ws/files/69281091/42003_2021_1809_MOESM3_ESM.mp4
UR - https://pure.uva.nl/ws/files/69281093/42003_2021_1809_MOESM4_ESM.mp4
UR - https://pure.uva.nl/ws/files/69281095/42003_2021_1809_MOESM5_ESM.xlsx
UR - https://pure.uva.nl/ws/files/69279861/42003_2021_1809_MOESM6_ESM.pdf
UR - http://www.scopus.com/inward/record.url?scp=85102686352&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=85102686352&partnerID=8YFLogxK
U2 - https://doi.org/10.1038/s42003-021-01809-8
DO - https://doi.org/10.1038/s42003-021-01809-8
M3 - Article
C2 - 33686188
SN - 2399-3642
VL - 4
SP - 306
JO - Communications Biology
JF - Communications Biology
IS - 1
M1 - 306
ER -