Figure S1 early log mid log late log stat.

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Julius Heath
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1 Figure S1 early log mid log late log stat. C D E F Fig. S1. The chromosome-expressed EI localizes to the cell poles independent of the growth phase and growth media. MG1655 (ptsi-mcherry) cells, which express the EI protein fused to mcherry from the chromosome, were grown in L () or in a minimal medium supplemented with glycerol (), glucose (C), lactose (D), mannitol (E) and ribose (F). Pictures were taken at different growth phases: early log, mid log, late log and stationary phase (stat.), as specified in the legend to Fig. 1. The EI-mCherry fusion protein was observed by fluorescence microscopy (red) and cells were observed with phase microscopy (grey). Shown are overlays of the signals from the fluorescence and phase microscopy (grey and red, merge). Scale bar corresponds to 1 µm.

Figure S2 EI-mCherry EI anti mcherry b. anti His b. 1 75 5 HPr-mCherry mcherry 37 1 2 3 4 5 1 2 3 4 5 glf-gfp C anti glf b. D anti GFP b.

Western blot analysis of the PTS and gl proteins. Immunoblot analysis was carried out as described in Supplementary Material and Methods.

Each panel is from a single blot and scan; removal of irrelevant lanes is marked by black separation lines.

Detection of EI and HPr proteins: Panels () and (), the general PTS proteins (specified in parenthesis hereafter) were expressed from the

(His-tagged mcherry); Lanes 4, pdllei-mcherry (His-tagged EI-mCherry); Lanes 5, pdllei-mcherry&hpr (His-tagged EI-mCherry and His-tagged HPr).

rbutin (1%) was added to the last 3 min of growth.

The results confirm that the mcherry-tagged EI and HPr are stable and the tag is not liberated (compare lanes 4 and 5 to lane 3).

2 Figure S2 EI-mCherry EI anti mcherry b. anti His b HPr-mCherry mcherry glf-gfp C anti glf b. D anti GFP b. 1 glf * 3 4* 5 6* 2* 3 5 glg-gfp E anti glg b. F anti GFP b. 5 glg * * 4 Fig. S2. Western blot analysis of the PTS and gl proteins. Immunoblot analysis was carried out as described in Supplementary Material and Methods. The primary antibodies that were used are specified above the panels. Each panel is from a single blot and scan; removal of irrelevant lanes is marked by black separation lines. star next to a lane number denotes that it shows the expression level of the relevant protein in our experiments, as detailed below. b., antibodies. Detection of EI and HPr proteins: Panels () and (), the general PTS proteins (specified in parenthesis hereafter) were expressed from the following strains or plasmids: lanes 1, MG1655 (ptsimcherry) (chromosome-encoded EI-mCherry); lanes 2, pd18 (vector only); lanes 3, pdllmcherry (His-tagged mcherry); Lanes 4, pdllei-mcherry (His-tagged EI-mCherry); Lanes 5, pdllei-mcherry&hpr (His-tagged EI-mCherry and His-tagged HPr). Expression of the plasmidencoded EI and HPr was induced by the addition of.1 % arabinose for 1 h. rbutin (1%) was added to the last 3 min of growth. Similar amounts of cell extracts were loaded, except for lanes 1, in which two times more extract was loaded. The results confirm that the mcherry-tagged EI and HPr are stable and the tag is not liberated (compare lanes 4 and 5 to lane 3). The ratio between the chromosomeencoded EI (panel, lane 1) and the plasmid encoded EI (panel, lane 4), after normalizing to the amount of extract loaded was.33 (i.e., three times more EI encoded from the plasmid than from the chromosome). The ratio between the plasmid-encoded HPr and EI proteins (panel, lane 5) was To calculate these ratios, the intensity of the bands was quantified by the MetaMorph software and normalized to the intensity in an area in the gel lacking detectable bands.

3 Fig. S2 (cont.) Detection of glf proteins: Panel (C), the gl proteins (specified in parenthesis hereafter) were expressed from the following strains or plasmids: lane 1, M1 (bgl + ) grown with salicin that induces expression of the bgl operon (chromosome-encoded gl proteins); lanes 2 & 3, MG1655 containing pdllhpr-mcherry&ei and pllk12f-gfp (glf-gfp) grown without (lane 3) or with (lane 4) IPTG; lanes 4 & 5, MG1655 (bgl - ) containing pdllhpr-mcherry&ei, pjs185 (glg-gfp) and pllk12f (glf) grown without (lane 4) or with (lane 5) IPTG; lane 6, MG1655 containing pdllhpr-mcherry&ei, pjs185 (glg-gfp) and pzlf (glf) grown with anhydrotetracycline hydrochloride (2 ng). Expression of the plasmid-encoded EI and HPr was induced by the addition of.1% arabinose for 1 h. rbutin (1%) was added to the last 3 min of growth for cells analyzed in lanes 2-6. Similar amounts of cell extracts were loaded, except for lanes 1, in which two and a half times more extract was loaded. Panel (D) shows the reaction of lanes 2 & 3 with anti-gfp antibodies. It is obvious that the level of the plasmid encoded glf that we used in the experiment shown in Fig. 7 (analyzed here in lane 4, no IPTG) is lower (hardly detectable without IPTG induction) than the native level encoded from the chromosome upon -glucoside induction (lane 1), even after taking into consideration that two and a half times more extract was loaded in the second case. The level of the plasmid-encoded glf-gfp that we used in the experiment shown in Fig. 3 (analyzed here in lane 2, no IPTG) was comparable to the native chromosome-encoded level (1.1 times higher after normalizing to the amount of extracts loaded). The level of the plasmid-encoded glf that we used in the experiment shown in Fig. S1 and S11 (analyzed here in lane 6) was significantly higher (over 1 fold) than the native chromosome-encoded level. Detection of glg proteins: Panel (E), the gl proteins (specified in parenthesis hereafter) were expressed from the following strains or plasmids: lane 1, M1 grown without -glucosides (no gl proteins expressed); lane 2, M1 grown with salicin, an inducer of the bgl operon (chromosomeencoded gl proteins); lane 3 & 4, MG1655 containing pdllhpr-mcherry&ei and pjs185 (glg-gfp) grown without (lane 3) or with (lane 4) IPTG. Expression of the plasmid-encoded EI and HPr was induced by the addition of.1 % arabinose for 1 h (lanes 3 & 4). rbutin (1%) was added to the last 3 min of growth for cells analyzed in lanes 3 & 4. In lanes 1 & 2, two and a half times more cell extract were loaded than in lanes 3 & 4. Notably, non-specific interactions with the polyclonal anti-glg antiserum due to cross-reacting antibodies are detected in all four lanes, independent of glg or glg-gfp expression. Panel (F) shows the reaction of the extracts in (E) with anti-gfp antibodies. It is obvious that the level of the plasmid-encoded glg-gfp, which we used in the experiments shown in all figures of this manuscript (lane 3, no IPTG) is lower (detectable with anti-gfp antibodies only after IPTG induction, lane 4) than the native level encoded from chromosome upon -glucoside induction (lane 2), even after taking into consideration that two and a half times more extract was loaded in the second case.

4 Figure S3 fluorescence intensity Fluorescence intensity of HPr-mCherry of Hpr-mCherry HPr-mCherry mcherry & & EI EI. HPr-mCherry mcherry & EI-N & EI-N average intensity at the poles average intensity in the cell 5 4 Ratio poles/cell ratio pole/cell Hpr-cherry HPr-mCherry & EI & EI. Hpr-cherry HPr-mCherry & EI-N & EI-N Fig. S3. Distribution of HPr in the cell in the presence of EI or EI-N. The fluorescent intensity of HPr-mCherry near the poles (blue) and its average intensity in MG1655 pts cells (purple), which express EI () or EI-N () together with the HPr-Cherry, were quantified for 2 cells, in each case, for the experiments shown in Fig. 2F and 4E, respectively. The intensities were determined by the MetaMorph software and normalized to the intensity in regions lacking cells in the respective fields. The ratio between the two values (yellow), shown in the lower panel, was 3.3 for () and 4.1 for ().

5 Figure S4 Phase HPr-GFP EI-mCherry merge C D E Fig. S4. EI and HPr localize to the cell poles independently, but the distribution of HPr in the cell is affected by EI and by PTS sugars. MG1655 pts (, D and E) or MG1655 (ptsi-mcherry) ( and C) cells, containing a plasmid that code for HPr-GFP (-C) or for both HPr-GFP and EI-mCherry (D-E) were grown in L (, D and E) or in minimal medium containing lactose () or mannitol (C) at 37 C (-D) or at 3 C (E). The GFP and mcherry fusion proteins were observed by fluorescence microscopy (green, GFP; red, mcherry) and cells were observed with phase microscopy. Overlays of the signals from the GFP and mcherry fluorescent signal (green and red, merge) are also shown.

6 Figure S5 II glc -GFP phase II glc -GFP EI-mCherry merge phase Fig. S5. The cytoplasmic protein II glc is evenly spread throughout the cell. MG1655 () and MG1655 (ptsi-mcherry) () cells, containing a plasmid which encodes II glc fused to GFP, were grown at 3 C in L (1 and 1) or at 37 C in minimal medium supplemented with either.4% glucose (2 and 2) or 2mM ribose (3 and 3). The II glc -GFP protein was expressed from pjpk12 () or pjp12 () without induction. The GFP and mcherry fusion proteins were observed by fluorescence microscopy (green, GFP; red, mcherry; green and red, merge) and cells were observed with phase microscopy (grey, phase). Scale bar corresponds to 1 µm.

7 Figure S6 Fluorescence intensity of glg-gfp glg-gfp expressed with: EI EI & DHPr average intensity at the pole average intensity in the cell Ratio pole/cell glg-gfp expressed with: EI EI & DHPr Fig. S6. Distribution of glg-gfp in the cell in the presence of one of the general PTS proteins or both. The fluorescent intensity of glg-gfp near the poles (blue) and its average intensity (purple) in MG1655 pts cells which express, besides glg-gfp, also EI () or EI and HPr (), were quantified for 2 cells, in each case, for the experiments shown in Fig. 5 and 5D, respectively. The intensities were determined by the MetaMorph software and normalized to the intensity in regions lacking cells in the respective fields. The ratio between the two values (yellow, shown in the lower panel) was 1.7 for () and 1.5 for ().

8 Fig. S7-I glg(h16y)- GFP phase merge Fig. S7-II glg(h28r)- GFP phase merge glg(h16y)- GFP mcherry merge glg(h28r)- GFP mcherry merge C EI C EI D HPr D HPr EI-mCherry +HPr E EI EI-mCherry +HPr E EI HPr-mCherry +EI F HPr HPr-mCheery +EI F HPr G EI(H189) G EI(H189) H HPr(H15) H HPr(H15) I EI-N) I EI-N EI-C Fig. S7. The residues required for glg phosphorylation are dispensable for its co-localization with the general PTS proteins at the poles. Images of MG1655 pts cells expressing glg(h16y)-gfp (Fig. S7-I) or glg(h28r)- GFP (Fig. S7-II). () Fluorescence images (green, left), phase contrast (grey, middle) and overlay (merge, right). (-I) Fluorescence microscopy images (green, GFP; red, mcherry; red and green, overlay) of the cells in () that contain also a second plasmid encoding EI-mCherry (), HPr-mCherry (C), EI-mCherry and HPr (D), HPr-mCherry and EI (E), EI(H189)-mCherry (F), HPr(H15)-mCherry (G), EI-N (H), EI-C (I). Cells were grown in rich medium at 3 C. Expression of the glg proteins was induced with IPTG (.1mM) for 1 hour. Scale bar corresponds to 1 µm. EI-C

9 Figure S8 [min] t t5 glg-gfp glg-gfp Fig. S8. glg migrates from the cell periphery to the cell poles after addition of the stimulating sugar. Shown are larger fragments from the fields presented in Fig. 7, before (t) and 5 min after (t5) arbutin addition.

10 Figure S9 Fluorescence intensity of glg-gfp fluorescence intensity of glg-gfp t t5 t t5 exp. 1 exp. 2 average intensity at the poles average intensity in the cell ratio pole/cell t t5 t t5 exp. 1 exp. 2 Fig. S9. glg migrates from the cell periphery to the cell poles after addition of the stimulating sugar. () The fluorescent intensity of glg-gfp near the poles (blue) and its average intensity in the cytoplasm and membrane (purple) of cells expressing glg-gfp together with glf and the general PTS proteins, before (t) and 5 min fter (t5) arbutin addition. The intensities were quantified for 2 cells from two independent experiments (exp. 1 and exp. 2), the first shown in Fig. 7, by the MetaMorph software and normalized to the intensity in regions lacking cells in the respective fields. Of note, quantifications were made for snapshot images, explaining the change in the absolute cell fluorescence between the time points. () The ratios (yellow) between each pair in () are as following: 1.9 (exp.1) or.97 (exp. 2) at t and 3.25 (exp.1) or 2.88 (exp.2) at t5.

glg is observed in foci that migrate from the cell periphery to the cell poles after addition

Fluorescence microscopy images of MG1655 pts cells grown in rich medium.

Expression of glf from pzlf was induced significantly by the addition of 2 ng/ml

11 Figure S1 [min] glg-gfp EI-mCherry t t5 t1 t2 Fig. S1. glg is observed in foci that migrate from the cell periphery to the cell poles after addition of the stimulating sugar. Fluorescence microscopy images of MG1655 pts cells grown in rich medium. Expression of glg-gfp was not induced. Expression of glf from pzlf was induced significantly by the addition of 2 ng/ml anhydrotetracycline hydrochloride (see Fig. S2C, lane 6 and figure legend), to slow glg-gfp release from glf and follow its route after sugar stimulation and before its accumulation at the poles Samples were taken before (t) and at the indicated times after the addition of 1% arbutin. The fluorescent signal of glg-gfp was twodimensional deconvolved to reveal the localization of most glg molecules.

12 Figure S11 fluorescence intensity t t2 t t2 snapshots average intensity at the poles time-lapse average intensity in the cell ratio pole/cell t t2 t t2 snapshots time-lapse Fig. S11. glg migrates from the cell periphery to the cell poles after addition of the stimulating sugar. The fluorescent intensity of glg-gfp near the poles (blue) and its average intensity (purple) in the cytoplasm and membrane of cells expressing glg-gfp together with glf and the general PTS proteins, before (t) and after (t2) arbutin addition, were quantified for 1 cells in snapshot images (see Fig. S1) and for 1 cells visualized by time-lapse microscopy. glf was overexpressed from pzlf as described in Fig. S1 (see explanation there). The intensities were determined by the MetaMorph software and normalized to the intensity in regions lacking cells in the respective fields. The ratio between the two values (yellow), shown in the lower panel, was 1.12 (snapshots) or 1.9 (time-lapse) at t compared to 2.91 (snapshots) or 1.99 (timelapse) at t2.

Fig. S1. CrgA intracellular levels in M. tuberculosis. Ten and twenty micrograms of

Supplementary data Fig. S1. CrgA intracellular levels in M. tuberculosis. Ten and twenty micrograms of cell free protein lysates from WT M. tuberculosis (Rv) together with various known concentrations