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.

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27Transmission electron microscopy (TEM) is an important imaging technique in bacterial 28 research and requires ultrathin sectioning of resin embedding of cell pellets. This method 29 consumes milli-to deciliters of culture and results in sections of randomly orientated cells. 30For rod-shaped bacteria, this makes it exceedingly difficult to find longitudinally cut cells, 31which precludes large-scale quantification of morphological phenotypes. Here, we 32 describe a new fixation method using either thin agarose layers or carbon-coated glass 33 surfaces that enables flat embedding of bacteria. This technique allows for the observation 34 of thousands of longitudinally cut rod-shaped cells per single section and requires only 35 microliter culture volumes. We successfully applied this technique to Gram-positive 36 Bacillus subtilis, Gram-negative Escherichia coli, the tuberculosis vaccine strain 37Mycobacterium bovis BCG, and the cell wall-lacking mycoplasma Acholeplasma 38 laidlawii. To assess the potential of the technique to quantify morphological phenotypes, 39we examined cellular changes induced by a panel of different antibiotics. Surprisingly, we 40 found that the ribosome inhibitor tetracycline causes significant deformations of the cell 41 membrane. Further investigations showed that the presence of tetracycline in the cell 42 membrane changes membrane organization and affects the peripheral membrane proteins 43 MinD, MinC, and MreB, which are important for regulation of cell division and elongation. 44Importantly, we could show that this effect is not the result of ribosome inhibition but is a 45 3 secondary antibacterial activity of tetracycline that has defied discovery for more than 50 46 years. 47Significance 48Bacterial antibiotic resistance is a serious public health problem and novel antibiotics are 49 urgently needed. Before a new antibiotic can be brought to the clinic, its antibacterial 50 mechanism needs to be elucidated. Transmission electron microscopy is an important tool 51 to investigate these mechanisms. We developed a flat embedding method that enables 52 examination of many more bacterial cells than classical protocols, enabling large-scale 53 quantification of phenotypic changes. Flat embedding can be adapted to most growth 54 conditions and microbial species and can be employed in a wide variety of microbiological 55 research fields. Using this technique, we show that even well-established antibiotics like 56 tetracycline can have unknown additional antibacterial activities, demonstrating how flat 57 embedding can contribute to finding new antibiotic mechanisms.58 59 60 Transmission electron microscopy (TEM) is a powerful tool to examine the morphology 61 and ultrastructure of bacterial cells. There are many bacterial embedding protocols for 62 TEM (1-5), but the basic procedure, i.e. embedding of cell pellets as small nuggets into 63 resin blocks, has not changed since the beginning of electron microscopy research on 64 bacteria 60 years ago (4, 6, 7). This technique has two major ...

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The electrochemical synthesis of ammonia is highly dependent on the coupling reaction between nitrate and water, for which an electrocatalyst with a multifunctional interface is anticipated to promote the deoxygenation and hydrogenation of nitrate with water. Herein, by engineering the surface of bimetallic Ni/Co-MOFs (NiCoBDC) with hydrogen-substituted graphdiyne (HsGDY), a hybrid nanoarray of NiCoBDC@HsGDY with a multifunctional interface has been achieved toward scaleup of the nitrate-to-ammonia conversion. On the one hand, a partial electron transfers from Ni 2+ to the coordinatively unsaturated Co 2+ on the surface of NiCoBDC, which not only promotes the deoxygenation of *NO 3 on Co 2+ but also activates the water-dissociation to *H on Ni 2+ . On the other hand, the conformal coated HsGDY facilitates both electrons and NO 3 − ions gathering on the interface between NiCoBDC and HsGDY, which moves forward the rate-determining step from the deoxygenation of *NO 3 to the hydrogenation of *N with both *H on Ni 2+ and *H 2 O on Co 2+ . As a result, such a NiCoBDC@ HsGDY nanoarray delivers high NH 3 yield rates with Faradaic efficiency above 90% over both wide potential and pH windows. When assembled into a galvanic Zn-NO 3 − battery, a power density of 3.66 mW cm −2 is achieved, suggesting its potential in the area of aqueous Zn-based batteries.