With ribose as substrate, growth rates are considerably improved,

With ribose as substrate, growth rates are considerably improved, but still not as high as with glucose, which is the preferred carbon source of B. subtilis (Fig. 3a; Singh et al., 2008). Samples were taken periodically and the phosphorylation state of Crh was analyzed (Fig. 3b). For comparison, the phosphorylation state of HPr was also determined (Fig. 3c). To discriminate between HPr(Ser~P) and HPr(His~P),

which migrate at the same position on the gel, a second aliquot of each sample was heated prior its loading onto the gel (Fig. 3c, even-numbered lanes). This leads to loss of the thermo-labile phospho-histidine bonds, whereas the serine-phosphate bonds are stable and remain intact. The comparison of both aliquots allows an estimation of the degree of phosphorylation of each site. During growth on the various substrates, the phosphorylation patterns of both Crh and HPr changed Epacadostat FK228 in a similar manner. Both proteins were detectable in their non-phosphorylated as well as serine-phosphorylated forms

during the exponential growth phase. As observed before (Fig. 2 and Singh et al., 2008), the ratio of the two forms depended on the carbon source (Fig. 3b, compare lanes 1, 4, 8; Fig. 3c, compare lanes 2, 8, 16). However, upon transition to the early stationary phase, the amount of Ser-phosphorylated Crh and HPr decreased drastically. When glucose was the carbon source, Crh as well as HPr was completely non-phosphorylated at Ser46 when cells entered the stationary growth phase (Fig. 3b, lane 3; Fig. 3c, lane 6). When succinate or ribose was the Gemcitabine carbon source, the extent of phosphorylation at Ser46 also decreased but a small amount of HPr(Ser)~P and Crh~P was detectable even upon entry into the stationary growth phase (Fig. 3b, lanes 7, 10; Fig. 3c, lanes 14, 20). The majority of phosphorylated HPr species detectable in this growth phase were phosphorylated at the His15 residue (Fig. 3c, compare lanes 5 and 6, lanes 13 and 14, lanes 19 and 20). There were no major changes in the total amounts of Crh or HPr under the various conditions (Fig. 3b and c, bottom panels). Finally, we wanted

to confirm that scarcity of the carbon source prevents phosphorylation of Crh and HPr at their Ser46 sites when cells enter the stationary growth phase. To this end, the wild-type strain was grown once again in minimal medium supplemented with glucose. After 7 h growth, i.e. the time of transition to the stationary growth phase, the culture was split and glucose was added to one of the two resulting cultures. The culture treated with additional glucose resumed growth and reached a final OD600 nm of 8.4, whereas the untreated culture entered the stationary growth phase, yielding a final OD600 nm of 3.7 (Fig. 4a), demonstrating that scarcity of the carbon source is growth-limiting under these conditions. Subsequently, the phosphorylation states of Crh and HPr were analyzed in samples that were taken periodically during growth (Fig. 4b).

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