B per extraction reagent

Bacteria were grown to an OD₆₀₀ ~0.3 and collected (8000 g, 5 min). For MtlA analysis, lysates were prepared by mixing ~10⁷ cells with SDS-loading buffer and heating at 95 °C for 10 min. For MtlR analysis, cell pellets from 5 to 50 ml cultures were lysed with B-PER Extraction Reagent (Thermo Fisher Scientific) in the presence of DNAse I, following the suggested protocol of the manufacturer; the lysate was then mixed with SDS-loading buffer. The proteins were resolved on 4–20 % TRIS gels (Bio-Rad) and transferred to nitrocellulose membranes. The membranes were treated with polyclonal anti-FLAG (abCam), polyclonal anti-HA (abCam), monoclonal anti-RpoB (abCam), or monoclonal anti-RNAPα (BioLegend) antibodies. Antibodies were detected using secondary antibodies with IR680 and IR800 dyes attached (Licor). As a membrane protein, MtlA has a tendency to oligomerize, even under the denaturing conditions of SDS-PAGE. Signals were visualized and band densities were measured using an Odyssey imager (Licor).

To every gram of bacterial cell pellet, 4ml of B-PER extraction reagent (Thermo Scientific, #78248) supplemented with DNAse I (Thermo Scientific, #90083) and protease cocktail (Thermo Scientific, #87785) was added. The suspension was incubated at 22°C for 60min with gentle shaking. Soluble and insoluble proteins were partitioned by centrifuging bacterial cell lysate at 15,000xg for 10 min at 4°C. Clear supernatant containing soluble proteins was passed through a 0.45μm membrane (Millipore, #HPWP04700) and used for immobilized-metal affinity chromatography (IMAC). Insoluble lysed bacterial biomass was resuspended to its original volume with Tris-buffered saline (TBS). Both, soluble and insoluble fractions were stored in small aliquots at -80°C for further analysis.