2.4. Recruitment of SgrT to the Membrane by EIICBGlc Can Be Visualized By in vivo Fluorescence Microscopy For further analysis of the interaction between SgrT and EIICBGlc we performed fluorescence microscopy to find out more about the distribution pattern of the two proteins in living cells. As shown in Figure 4, plasmid

encoded EIICBGlc tagged with Gfp was homogeneously distributed in the cytoplasmic membrane (4B), whereas SgrT tagged with Gfp could be detected in an EIICBGlc-negative #selleck chemicals Dorsomorphin keyword# strain only in the cytosol (4D). In contrast, in E. coli ptsG+ cells that were grown in the presence of glucose, the localization of SgrT-Gfp clearly shifted to the membrane, which indicates a sequestration of SgrT by unphosphorylated Inhibitors,research,lifescience,medical EIICBGlc (4F). In accordance with the previously obtained results of the crosslinking experiments, EIICBGlcP384R, unlike the wild

type protein, was not capable of sequestering SgrT-Gfp (4H), which, yet again, indicates the missing interaction between the two proteins. Figure 4 Fluorescence microscopy for the determination of EIICBGlc and SgrT localization. Bright field (upper lane) and fluorescence microscopy (lower lane) were performed with three different strains Inhibitors,research,lifescience,medical expressing EIICBGlc or SgrT derivatives tagged with Gfp. A and B: JKA12 (ΔptsG::cat ΔsgrRST::neo) expressing EIICBGlc-Gfp; C and D: JKA12 expressing SgrT-Gfp; E and F: JKA1 (ptsG+ΔsgrRST::neo) expressing SgrT-Gfp. G and H: JKA18 (ptsGP384RΔsgrRST::neo) Inhibitors,research,lifescience,medical expressing EIICBGlcP384R and SgrT-Gfp. All cells were grown in minimal medium with 0.2% glucose. These results indicate a sequestration of SgrT by unphosphorylated EIICBGlc (wild type), but not by EIICBGlcP384R in living cells. 2.5. Discussion

Overflow metabolism, which is accompanied in E. coli by acetate production, is a metabolic phenomenon which takes place when the rates of carbohydrate Inhibitors,research,lifescience,medical transport and glycolysis exceed a critical value due to high growth rates under aerobic growth conditions [32]. Acetate is produced from acetyl-CoA via acetyl-phosphate. Thus, under conditions of high glycolytic flux overflow metabolism directs a portion of the excess acetyl-CoA to acetate production. In addition, other byproducts such as selleck kinase inhibitor succinate, lactate, pyruvate, or methylglyoxalate can also be produced under these conditions. During overflow metabolism, Dacomitinib not all of the substrate is converted into biomass which constitutes an enormous disadvantage for biotechnological processes. Accordingly, the phenomenon of overflow metabolism has been investigated in greater depth during the past years in an effort to make industrial biotechnology more cost-efficient and economically advantageous [33]. The preferred carbon source in biotechnological applications is glucose, which in E.