The various substrates form a hierarchy in their ability to trigger phosphorylation of HPr at Ser46 (Singh et al., 2008). Fructose-1,6-bisphosphate (FBP) has been identified as the main factor that allosterically activates kinase activity of HPrK/P, but other metabolites may also play a role (Jault et al.,

2000; Ramström et al., 2003). Bacillus subtilis and other Bacilli possess carbon-flux-regulating histidine protein (Crh), which shares over 40% sequence identity with HPr (Galinier et al., 1997). Crh lacks His15, but contains Ser46 and accordingly it becomes (de)phosphorylated by HPrK/P in vitro (Lavergne et al., 2002). Crh~P can likewise form a complex with CcpA and contributes to CCR, but to a weaker extent than HPr(Ser)~P. Hence, Crh~P can only partially replace HPr(Ser)~P in CCR (Galinier et al., 1997; Singh et al., Regorafenib research buy 2008). This weaker contribution of Crh~P to CCR can be ascribed to its much lower levels in the cell and its lower binding affinity for CcpA as compared with HPr(Ser~P) (Görke Selleckchem ABT888 et al., 2004; Seidel et al., 2005). Therefore, Crh was regarded for a long time as back-up factor for CCR. However, recently a distinct role for Crh has been identified. It was found that non-phosphorylated Crh binds to and inhibits activity of the metabolic enzyme methylglyoxal synthase, MgsA,

in B. subtilis (Landmann et al., 2011). MgsA catalyzes the formation of methylglyoxal from dihydroxyacetone-phosphate, initiating a glycolytic bypass. This pathway may relieve cells from Epothilone B (EPO906, Patupilone) sugar-phosphate stress, when carbohydrate uptake rates exceed the capacity of the lower branch of the Embden–Meyerhof–Parnas (EMP) pathway (Weber et al., 2005). To understand the physiological conditions under which Crh exerts its regulatory functions, it is crucial to know its phosphorylation state in vivo. Indirect evidence from studies on CCR suggested that the phosphorylation of Crh and HPr at their Ser46 sites has similar dynamics (Galinier et al., 1997; Singh et al., 2008). Direct proof of this hypothesis has so far been hindered technically by the low cellular abundance of Crh (Görke et al., 2004). This might explain why Crh~P was detected in only one of several phosphoproteome

studies (Eymann et al., 2007). In the present work, we overcame these limitations and analyzed phosphorylation of Crh in vivo in response to different nutritional conditions. A direct method was used that involves separation of Crh and Crh~P in cell extracts by non-denaturing gel electrophoresis. Crh was detected using a tailor-made sensitive antiserum specifically directed against a C-terminal peptide of Crh. Bacillus subtilis strains were grown at 37 °C in CSE minimal medium (C-medium supplemented with sodium succinate and potassium glutamate; Martin-Verstraete et al., 1995) supplemented with 50 mg L−1 tryptophan and 0.5% of the indicated carbon source. The strains used were B. subtilis 168 (trpC2; wild-type), QB7097 (trpC2 Δcrh::spec; Singh et al.