In order to compare growth kinetics basic medium (BM) composed of 1% casein peptone, 0.5% yeast extract, 0.5% NaCl,
0.1% K2HPO4 × 3 H20, and 0.1% glucose was inoculated with bacterial over-night cultures grown in tryptic soy broth (TSB; Fluka) at an OD578 of 0.08 and cultivated either with aeration (50 ml in notched 100 ml flasks on a shaker) or without (completely filled, sealed 15 ml tubes) at 37°C and OD578 was measured at several time points. Cultures of the complemented mutant were supplemented with 10 μg/ml chloramphenicol. To compare capacities to catabolize Copanlisib various substrates the various strains were used to inoculate ApiStaph tubes (BioMérieux), which were incubated and evaluated according to the manufacturers’ manual. Extracellular metabolome analysis by 1H-NMR For quantification of extracellular metabolites TSB overnight cultures of RN4220 wild type and the Δfmt mutant were used to inoculate 100 ml Iscove’s modified Dulbecco’s media (IMDM) without phenol red (Gibco) in notched 250 ml flasks at an OD578 of 0.1. The cultures were incubated on a shaker at 37°C. Samples were taken at 8 h and 24 h to determine the OD578 and
obtain culture supernatants by centrifugation with subsequent filtration (0.22 μm pore size). Samples were prepared and analyzed Selleck Vistusertib by 1H-NMR as described recently [21, 22]. Briefly, 400 μl of supernatants were mixed with 200 μl phosphate buffer (0.2 M; pH 7.0) and applied to a Bruker®Avance II 600 MHz spectrometer operating with TOPSPIN 2.0 (Bruker®Ricolinostat nmr Biospin). Metabolites were identified by comparison with pure reference compound spectra. Trimethylsilylpropionic acid d4 was used as internal standard. All spectra were processed in Chenomx NMR Suite 4.6 (Chenomx, Edmonton, AB, Canada) and selected metabolites were quantified by computer-assisted manual fitting of metabolite peaks. RNA isolation and microarray analyses To compare the transcription profiles Etomidate of the RN4220 wild type and Δfmt mutant the strains were grown in BM (13 ml in notched 50 ml flasks) at 37°C to an OD578 1.0 under aerobic conditions or to an OD578 0.5 under anaerobic conditions (completely filled
and sealed 15 ml tubes). Bacteria were harvested via centrifugation and immediately frozen at −80°C. Samples were then thawed on ice and resuspended with 1 ml Trizol (Invitrogen) to inhibit RNases and bacteria were disrupted with 0.5 ml glass bead suspension in a homogenizer. The supernatants of these lysates were mixed with 200 μl chloroform for 60 s and incubated for another three minutes to extract the RNA. After centrifugation (15 min; 12,000 × g; 4°C) the upper phase was collected and pipetted into 500 μl isopropanole. After 10 min at room temperature the samples were centrifuged for 30 min again to collect supernatants. Then 500 μl 70% ethanol was added and the samples were centrifuged at 4°C, 7,500 × g for 5 min.