7 Batch; 5.6 mM Glc 99.7 98.9 check details 99.8 Chemostat, D = 0.15 h-1; 0.56 mM Ac 93.9 71.4 90.1 Batch; 0.56 mM Ac 92.1 76.0 94.1 Chemostat, D = 0.15 h-1; 5.6 mM Ac 98.4 84.9 96.3 Batch; 5.6 mM Ac 94.6 83.2 96.6 Bhemostat, D = 0.15 h-1; 2.8 mM Glc, 2.8 mM Ac 99.0 97.2 93.5 Batch; 2.8 mM Glc, 2.8 mM Ac 99.8 99.5 99.8 Chemostat, D = 0.15 h-1; 0.28 mM Glc, 0.28 mM Ac 99.5 91.9 92.8 Batch; 0.28 mM Glc, 0.28 mM Ac 99.1 99.3 99.6 Overall, these results suggest that the promoter for mglBAC is expressed above background in a higher fraction of the population than the promoter for ptsG, and differences in ptsG expression between genetically identical

cells could be an indication of glucose uptake heterogeneity within clonal populations. Next, we used direct measurements of uptake to analyze the activity of the glucose-PTS transporter and to compare the transporter activity with the expression of PptsG-gfp. 2-NBDG, 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxy-D-glucose, is a fluorescent D-glucose analog, and has been used to study the dynamics of glucose uptake via the phosphotransferase system (PTS) in single cells of E. coli[18, 34]. Since 2-NBDG is exclusively taken up via Glc-PTS, cells will fluoresce only if their PTS system is active and the glucose analog is transported inside the cell. As this assay uses a glucose analog that cannot be metabolized,

the results can be interpreted only in the context of the activity of the transport Bucladesine system and not as a general measure Acetophenone of metabolic activity of a cell. Our data indicate that not all cells use the PTS system to take up glucose from the media (Figure  2, medium supplemented with 0.56 mM Glc). How do the rest of the cells take up glucose – do they maybe employ alternative carbon sources? There are two possibilities.

First, cells might use Mgl or another glucose transporters. Second, it is possible that the cells use excreted acetate as (an additional) carbon source. We also found that even if the PptsG-gfp reporter strain fluoresces, it does not necessarily mean that PTS is actively transporting glucose (Figure  2). This became evident in control experiments where we grew cells in medium containing acetate or arabinose as the sole carbon source. Around 80% of the gated population growing in acetate (around 60% growing in arabinose) expressed the ptsG reporter above the background level, without any glucose present to induce the expression or to be transported (Additional file 1: File S1). Furthermore, in these conditions the PptsG-gfp reporter showed a high degree of variation in expression (Figure  2). Figure 2 Comparison of Glc-PTS activity and PptsG- gfp expression in different chemostat conditions. The distributions show Glc-PTS (PtsG/Crr) activity (orange) based on uptake of a fluorescent glucose analog, expression of PptsG-gfp (green) and negative control (wild-type MG1655, black).