Figure 4. Glutamine inhibits PTS^Ntr phosphorylation.

(a) Glutamine inhibits autophosphorylation of EI^Ntr but not EI^Ntr_L83Q. Autophosphorylation assays of EI^Ntr and EI^Ntr_L83Q using [³²P]PEP as a phosphoryl donor in the absence or presence of increasing concentration of glutamine (0, 2, 5, 10 mM). The full autoradiography is available in Supplementary Fig. 8a. (b) The EI^Ntr-dependent phosphorylation of EIIA^Ntr is enhanced upon nitrogen starvation. In vivo phosphorylation assays of EIIA^Ntr in −N or +N conditions supplemented with (+Xyl) or without (−Xyl) xylose in wild-type (WT) or ΔptsP cells expressing 3FLAG-ptsN from PxylX promoter. Error bars=s.d.; n=3. Statistically significant differences by Student's t-test are indicated as **P<0.01% (n=3). The full autoradiography is available in Supplementary Fig. 8b. (c) Immunoblotting of protein samples extracted from WT and ΔptsP cells expressing 3FLAG-ptsN from PxylX promoter, incubated 3 h in M5GG supplemented with (+Xyl) or without (−Xyl) xylose. MreB was detected in all conditions, while 3FLAG-EIIA^Ntr was detected only in the presence of xylose. The full blot is available in Supplementary Fig. 8c.