Typhoon 9200

Five culture tubes were set up to have 1.95 mL of LB, followed by the addition of 50 µL of 10⁻³ to 10⁻⁷E. coli stocks (which contained 10⁵ to 10 cells, respectively). These tubes were then placed in a 37°C incubator (with shaking). 50 µL of culture from each well was taken at 0, 1, 4, 8, and 12 h and subjected to ultrasonic treatment to stop cell growth. Thus treated culture was placed in a well of a 96-well plate containing 50 µL of 2× RB with 2 pmol of RFD-EC1. After incubation for 30 min, the fluorescence intensities of the reaction wells were taken using Typhoon 9200, variable mode (GE Healthcare. The data is presented in Fig. 2C.

Labeled OGs (25 and 50 μg) or unlabeled OGs (25 μg) were added to loading buffer solution (TBE 1X, 4% glycerol, 0.1% bromophenol blue). The samples were loaded on a polyacrylamide gel (15% acrylamide-bis acrylamide 29:1, TBE 20X, 0.1% APS, 0.16% TEMED) and run at constant voltage of 100 V for 20 min. Gel images were acquired using a Typhoon 9200 laser scanner (Ge-Healthcare) and the following scan settings: excitation wavelength of 480 nm and emission wavelength of 520 nm. Next, the gel was stained with Ruthenium Red (0.02%, w/v) and image was acquired with a GelDoc (BIO-RAD) (Figures 1A,B).

Measurements of RSH_Mex1-352 enzyme kinetics were carried out as described above but with 80 nM protein. Reactions were carried out with 8 mM CoCl₂ for 2 h. End-point assays were used since using continuous assays was impractical in this case due to low method sensitivity in detecting low (p)ppNpp amounts. The values obtained should be taken with caution and thus are referred to as apparent Km. The apparent Km for GTP was measured with ATP at 8 mM and titrated GTP (1–8 mM). For the apparent Km for ATP, GTP was held at 8 mM and ATP varied from 0.25 to 2 mM. The samples were processed as described above and TLC was run in 1.5 M KH₂PO₄ (pH 3.4) buffer. Autoradiograms were visualized using a phosphorimager (Typhoon 9200, GE Healthcare). Spots corresponding to pppGpp were quantitated using a UVP Visionworks software and the data was analyzed with GraphPad Prism 5; apparent Km values were obtained from the fit to the following equation: V = [Vmax (S)] ÷ [Km + (S)].

The measurement of cellular guanosine tetra-phosphate (ppGpp) level was based on the [³²P]-labeling of nucleotides method developed by thin-layer chromatography on PEI-cellulose plates [72 (link)]. Fresh V. cholerae culture was suspended in PBS to obtain OD₆₀₀ = 1.0 on the McFarland scale as measured by a Densila-Meter II (ErbaLachema, Brno, Czech Republic). The suspension was diluted at a 1:10 ratio in a minimal MOPS medium containing 0.4 mM KH₂PO₄ (0.2% casamino acids). Then, the [³²P]-orthophosphoric acid was added to the cultures to a final concentration of 150 μCi/mL, and bacteria were grown at 37 °C for 1 h with shaking 165 rpm/min. After this time, the bacterial cultures (150 μL) were transferred to wells in a 96-well plate. SFN, PEITC in a range of concentrations were added. The amino acid starvation was induced by adding an L-serine amino acid- serine hydroxamate (SHX) analogue to a final concentration of 0.8 mg/mL to the bacterial cultures. Samples were collected at the indicated time, then extracted with formic acid (13 M) in two cycles of freezing and thawing in liquid nitrogen. Samples were centrifuged (5000× g, 5 min), and the nucleotides present in the supernatant were separated by thin layer chromatography on a polyethyleneimine (PEI) TLC plate with cellulose in 1.5 M potassium phosphate buffer and analyzed with Typhoon 9200 (GE Healthcare, Sweden).

Absorbance measurements were recorded with a microplate reader (Tecan infinite M1000 Pro, Männedorf, Switzerland). The autoradiogram and fluorescent images of gels were obtained using Typhoon 9200 variable mode imager (GE Healthcare) and Bio-Rad image system. The intensity of each band was analyzed using Image Quant software (Molecular Dynamics). Thermal melting temperatures were measured by Roche LightCycler96 quantitative fluorescence PCR instrument. PT times were measured by using a semi-automatic coagulometer (Ruimai, Nanjing, China).

Fluorescence scans were obtained using a variable mode imager (Typhoon 9200; GE Healthcare).