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Mezhyrova et al. Microbiome Res Rep 2023;2:28 https://dx.doi.org/10.20517/mrr.2023.28 Page 5 of 17

Liquid bead ion desorption mass spectrometry
LILBID is a native mass spectrometry ionization technique in which ion release results from irradiating
[25]
small microdroplets (Ø 30 nm) containing the analyte of interest with an IR laser . The microdroplets are
generated with a commercially available piezo-driven droplet generator (MD-K-130; Microdrop
Technologies GmbH, Norderstedt, Germany). Droplet irradiation occurs at a frequency of 10 Hz and the IR
laser operates at the absorption wavelength of water (2.94 µm). The laser energy can be varied between a
range of 10 and 23 mJ. Latter enables the study of intact as well as partially dissociated non-covalently
bound protein/protein complexes. A homebuilt time-of-flight setup including a reflectron operating at high
vacuum conditions (10 mbar) is used as an analyzer. For more details regarding LILBID-MS, please see .
[26]
6
A solution of 50 mM ammonium acetate (pH 7.4) was used as buffer for LILBID-MS analysis of full-length
MS2-L and truncated constructs in NDs. For analysis of detergent-solubilized MS2L derivatives, a buffer of
50 mM ammonium acetate (pH 7.4) supplemented with 0.02% Brij78 was used. Buffer exchange was
performed with the help of Zeba Micro Spin desalting columns (Thermo Scientific, USA, 7 kDa MWCO). A
sample volume of 4 µL was used for each measurement. Ion detection was performed in negative ion mode
and all shown mass spectra are normalized to 1 and represent averaged signals of 1,000 droplets. Massign
was used for spectra and data processing .
[27]

DnaJ pulldown
Each pulldown reaction contained 60 µL of a 5% magnetic bead suspension (MagStrep “type3” XT beads,
iba Lifesciences). The beads were washed 3 times with 1 mL ddH2O and 3× with 1 mL StrepII buffer. If
detergent-solubilized proteins were utilized, the working buffer was supplemented with 0.02% Brij78. 50 µg
of purified, StrepII-tagged bait proteins (MS2-L derivatives) were immobilized by incubating them with the
beads in StrepII buffer (total volume 500 µL) for 1 h at 4 °C in an overhead rotor device. To remove
unbound bait, the beads were washed 3× with 1 mL of Strep buffer. Subsequently, a defined amount of the
prey protein DnaJ (50 µg) was added and incubated for 4 h at 4 °C in an overhead rotor device for binding.
Finally, unbound prey was removed by washing 3× with 1 mL of Strep buffer and the protein complexes
were eluted by adding 40 µL of SDS loading buffer and heating at 75 °C for 10 min.

In vivo expression of MS2-L derivatives
Plasmids containing toxin constructs were transformed into Lemo21 (DE3) or T7 Express cells. The
transformed cells were plated on LB-agar plates containing the appropriate antibiotic and incubated
overnight at 37 °C. To minimize the accumulation of escape mutations against toxins, which could possibly
be synthesized by background expression, bacterial cultures (200 mL LB medium with the appropriate
antibiotic or a combination thereof) were inoculated directly from the agar plates the next morning. The
cultures were incubated at 37 °C and 180 rpm until an OD of 0.2-0.3 and toxin synthesis was then induced
600
with 1 mM IPTG. Bacterial growth was monitored by periodic OD measurements. For subsequent LSM
600
imaging, 2 mL bacterial samples were taken at the indicated time points, centrifuged (5,000 × g, 4 °C, 2 min),
resuspended in 100 µL of 10 mM Tris-HCl, pH 8.0, and flash frozen in liquid nitrogen. Samples were stored
at -80 °C until further use.

Confocal laser scanning microscopy
To capture living bacterial cells, agarose pads (1% agarose in 89 mM Tris-borate, 2 mM EDTA, pH 8.0) were
prepared and arranged on glass slides. For bacterial viability assays, the LIVE/DEAD BacLight Bacterial
TM
TM
Viability Kit (ThermoFisher Scientific) was used. A dilution of the propidium iodide (PI) dye (0.3 µL PI +
100 µL PBS) was prepared and 0.5 µL of it was added to 15 µL of the thawed bacterial culture. Cells were
then incubated for 15 min at RT under light exclusion. Subsequently, the cells were centrifuged (14,000 × g,
RT, 2 min), the supernatant was discarded and the cells were washed once with 100 µL PBS. After another
centrifugation step at the previously described conditions, cells were resuspended in 15 µL PBS. 3 μL of