An investigation into the physiological roles of NAD+-GDH enzyme in M. bovis is currently underway. Methods Bacterial strains and culture methods Mycobacterium smegmatis MC155 2 was routinely selleck compound cultured in 7H9 medium (Difco) supplemented with 10% Oleic acid-Albumin-Dextrose-Catalase enrichment (OADC; Middlebrook) until an OD600 of approximately

0.8. The bacteria were transferred to Kirchner’s minimal medium [57] in which asparagine was replaced with ammonium sulphate ((NH4)2SO4) as the sole nitrogen source. It has previously been shown that an increase in NH4 + concentration from 3.8 mM to 38 mM caused a 10-fold reduction in M. tuberculosis activity [23]. The observed response of GS activity to the change in NH4 + concentration is indicative that bacteria exposed to 3.8 mM NH4 + were starved

of nitrogen. In addition to a change in activity, a response in the level of GS transcription was also observed [47]. An (NH4)2SO4 concentration of 3 mM was thus used to induce nitrogen starvation in M. smegmatis whereas Kirchner’s medium containing 60 mM (NH4)2SO4 Dibutyryl-cAMP solubility dmso was considered as nitrogen sufficiency or excess. M. smegmatis liquid cultures were maintained at 37°C with shaking. Preparation of crude protein extract M. smegmatis was harvested by centrifugation and resuspended in 1 ml of Tris-HCl (pH 8) or phosphate buffer (Na2H2PO4/K2HPO4; pH 7.0). The cells were disrupted by ribolysing at maximum speed for 20 sec (Fastprep FP120, Bio101 Savant) and immediately placed on ice for 1 min thereafter. This ribolysing procedure was repeated 3 to 4 times with intermittent cooling on ice. The sample was centrifuged at 4°C in a benchtop

centrifuge (Mikro 200, Hettich Zentrifugen) to remove insoluble material and the total protein concentration was determined using the Bradford assay (Bio-Rad, Germany) according to the manufacturer’s instructions. Enzyme assays Glutamate Bacterial neuraminidase dehydrogenase activity assays i) NADPH-specific Glutamate dehydrogenase selleck chemicals llc NADPH-GDH activity was assayed essentially as described by Sarada et al. [28]. The NADPH-GDH forward reaction (reductive aminating activity) was assayed by preparation of a 1 ml reaction system containing 100 mM Tris HCl (pH 8.0), 100 mM NH4Cl; 10 mM α-ketoglutarate and 0.1 mM NADPH. The NADPH-GDH reverse reaction (oxidative deaminating activity) assay preparation consisted of 100 mM Tris-HCl (pH 9.0); 200 mM glutamate and 0.1 mM NADP+. The reactions were initiated by the addition of 10 μg M. smegmatis crude protein extract. ii) NADH-specific GDH The activity of both the forward and reverse NADH-GDH reactions were assayed using a combination of methods from Loyola-Vargas et al. [56] and Miñambres et al.[18]. The 1 ml NADH-GDH forward reaction (reductive amination) assay consisted of 100 mM Phosphate buffer (HK2PO4/H2NaPO4; pH 7.