The MG1655 (wild-type K-12) strain and the CF5802 strain [25], deficient in polyphosphate kinase and exopolyphosphatase (MG1655 Δppk-ppx::km) were gifts from Dr. M. Cashel (Laboratory of Molecular Genetics, NICHD, National Institutes of Health, Bethesda, MD, USA). The heat-sensitive CV2 (CGSC # 4682, initially derived from E. coli strain K-10) [26] and the SpoT-deficient NF161 (CGSC # 5244 derived from K-12) [12] strains were obtained from the E. coli Genetic Resource Center (Yale

University, New Haven, CT, USA). A strain devoid of RelA (MFT702 ΔrelA derived from MG1655) [10] was a gift from Dr. T. Conway (Advanced Center for Genome Technology, University of Oklahoma, Norman, OK, check details USA). The BL21 strains overexpressing either human recombinant ThTPase as GST fusion protein (BL21-hThTPase) or E. coli adenylate kinase (BL21-AK) were produced as previously described [21, 27]. Growth and processing of the bacteria The bacteria were grown overnight (37°C, 250 rpm) in 50-100 mL Luria-Bertani (LB) medium (S3I-201 ic50 tryptone, 10 g/L; yeast extract, 5 g/L; NaCl, 10 g/L, pH 7.0). The bacteria were centrifuged (5 min; 5000 × g) and suspended in the initial volume of M9 minimal medium (Na2HPO4, 6 g/L; KH2PO4, 3 g/L; NaCl, 0.5 g/L; NH4Cl, 1 g/L; CaCl2, 3 mg/L; MgSO4, 1 mM, pH 7.0) containing various metabolic

substrates in sterile PS-tubes (18,0/95 mm, 14 mL, Greiner Bio-One BVBA/SPRL, Wemmel, Belgium). If not otherwise stated, the bacteria Celastrol were incubated at 37°C with shaking KU-60019 price (250 rpm). The density of the cultures was determined by reading the absorbance at 600 nm (A600). After incubation, the bacteria were sedimented as above, the pellets were suspended in 12% TCA, the precipitated proteins were spun down (15 min, 15,000 × g) and the pellet was dissolved in 0.8 N NaOH for protein determination by the method of Peterson [28]. The supernatant was treated with diethyl ether to remove TCA and analyzed by HPLC for thiamine compounds [29]. For the determination of adenine nucleotides by HPLC, TCA (12%) was added directly to the bacterial suspension. For growth in the absence of oxygen,

the bacteria were incubated in sterile tubes with screw caps (Greiner Bio-One BVBA/SPRL, Wemmel, Belgium). The culture was sparged with N2 for 1 min and the tubes were hermetically closed before incubation. Determination of thiamine compounds and adenine nucleotides Thiamine compounds were determined by HPLC as previously described, after conversion to fluorescent thiochromes [29] and ATP was determined by luciferin luminescence using the Bac-Titer-Glo kits (Promega Benelux b.v., Leiden, The Netherlands). For determination of the energy charge [20], ATP, ADP and AMP concentrations were determined by a HPLC method, using fluorescence detection after ethenylation with chloroacetaldehyde [30]. Intracellular concentrations were estimated assuming an intracellular volume of 3.2 μL per mg of protein [8].