The second, refolding step included washing with AZD1208 cell line BB at linearly decreasing urea concentrations (from 8 to 0 M). The protein was eluted with a linear gradient of imidazole from 5 to 500 mM. Protein was collected
at 0–250 mM imidazole concentrations in a total volume of 4–5 mL. Rpf-containing fractions (30–50 μg mL−1) were dialyzed against 50 mM citric acid–sodium citrate buffer (pH 6.0). Protein samples were stored at +4 °C for 1 week without a significant loss in its activity. Myñobacterium smegmatis strains were grown under the conditions that favored the entering wild-type strain to ‘nonculturable’ (NC) state (inability to produce colonies on solid media) in stationary phase after cultivation of mycobacteria in the modified Hartman-de-Bont medium, lacking K+, at 37 °C for 120 h under aeration (Shleeva et al., 2004). In the other model, the strains under study were incubated for 4.5 months after growth in N-limited SR-1 medium to produce morphologically distinct ovoid cells (Anuchin et al., 2009). The ability of ‘NC’ cells to resuscitate in liquid medium was estimated using the most probable number (MPN) assays in triplicate repeats with inoculation of 0.1 mL cell suspensions to 0.9 mL of the modified Sauton medium in plastic 48-well microplates (Corning) as described previously (Downing et al., 2005). The Sauton medium that served for resuscitation contained (L−1): KH2PO4, 0.25 g; MgSO4·7H2O, 0.25 g; l-asparagine,
2 g; glycerol,
AUY-922 manufacturer 6 mL; ferric ammonium citrate, 0.025 g; sodium citrate, 1 g; 1% ZnSO4, 0.05 mL; ± recombinant RpfSm protein, 5 μg (pH 7.0). Cell suspensions were examined under a microscope Eclipse E4000 (Nikon, Japan) in the phase-contrast and epifluorescence modes after staining with propidium iodide (3 μM) to detect injured/dead cells or with 4′-6-diamidino-2-phenylindole (DAPI) (2 μg mL−1) bound to double-helix DNA. Excitation was at 510 and 330 nm, and emission was at >560 and >380 nm for propidium iodide and DAPI, respectively. One-milliliter aliquots were taken from stationary-phase (48 h) cultures in NB medium or from cultures stored for 4.5 months in N-limited SR-1 medium and were transferred into Petri dishes with 4 mL of liquid NB medium and then subjected to UV irradiation (BUV-30 lamp, 254 nm) as described elsewhere (Vorobjeva et al., 1995). Samples from the same Tacrolimus (FK506) cultures were also heated at 60–80 °C for 10 min. Cells after UV or heat treatment were plated onto solid NB medium for CFU assays. As already demonstrated, after cultivation for 68–70 h in the modified Hartman-de-Bont medium without K+ sources, stationary-phase wild-type M. smegmatis cells entered a dormant NC state and lost the ability to form colonies on the nutrient agar. NC cells of the wild-type strain were resuscitated in a liquid medium supplemented with Rpf. Similarly, the isogenic strain (Wt∷rpf) that harbors a plasmid containing the M.