Article Figures & Data
Figures
- Figure 1. Chemical structure of the maltodextrin compounds studied in this work.
The moieties corresponding to maltotriose of Cpd-1 and maltohexaose of Cpd-2 are boxed.
- Figure S1. Characteristics of E. coli strains used in this study.
(A) Characteristics of E. coli strains used in this study. The strains and plasmids were built and generously donated by the Misra's lab (33, 39). (B) Detection of the presence of the major porins, OmpC, and OmpF, and the maltoporin LamB in various strains grown in LB with or without 0.2% arabinose (Ara, induction of the LamB expression) by Western blots.
- Figure 2. Accumulation of Cpd-1 and Cpd-2 depends on LamB expression.
The strains were grown in different media to control the expression of the LamB porin and the MalE transporter; 2 components of the maltose regulon (see Figs S2 and S3). See Fig S1 for the characteristics and the corresponding immunoblots of the strains. (A) Presence of LamB and MalE by Western blot in RAM1292 and RAM2808 under different growth conditions. (B, C) Number of Cpd-1 (B) and Cpd-2 (C) molecules accumulated per cell in the various studied strains following analysis by spectrofluorimetry. The columns with bars (SDs) correspond to measurements carried out in triplicate. Calibration curves were used to obtain the number of molecules per cell (Fig S4). (D) Microfluorimetric images obtained with DUV microscopy with pellets of RAM2808 with or without induction of the LamB porin incubated without and with Cpd-1 or Cpd-2. Controls are RAM2808 cells incubated without Cpd-1 and Cpd-2. (E) Microfluorimetric results obtained from (D). Data are represented with a box-and-whisker plots, which is a way of summarizing the essential profile of a quantitative statistical series: the boxes represent data-points from the 25th to 75th percentiles; the middle horizontal lines represent the median data point and the whiskers show the span of the data for each sample. The outliers are represented by red + signs.
- Figure S2. Schematic representation of Mal transport and regulon.
(A) Maltodextrin transport across the outer and inner membranes via LamB (maltoporin) in the outer membrane, MalE (maltose-binding protein) in the periplasm and the maltose inner membrane transporters (MalF, G, K). Once in the cytoplasm, maltodextrin is metabolized by enzymes (MalP, Q, Z). (B) Organisation of mal genes and their regulation: crp (cAMP-binding protein, needed for the transcription of malT and the transport gene cluster) induces the expression of MalT (a transcriptional activator) which is then activated by maltotriose. Activated MalT (Ta) and also crp regulate the expression of the maltose operons. For our study, note that LamB and MalE are not co-transcribed in the same direction. The knock-out of LamB does not disturb the expression of MalE. mal genes involved in maltose transport are written in blue and mal genes involved in maltose degradation are written in green (23, 48).
- Figure S3. Detections of LamB and MalE.
Presence of LamB and MalE in RAM1292 cells grown with 0.4% glucose (RAM1292 Glc, repression of the maltose operon) or maltose (RAM1292 Malt, induction of the maltose operon); and RAM2808 grown with 0.4% maltose without or with 0.2% arabinose (RAM2808 Malt and RAM2808 Malt Ara, induction of the maltose operon and LamB expression). Maltose can induce the production of MalE in both strains (only the lamB gene is knocked out in the RAM2808 strain, lamB is provided on a plasmid in this strain). The first and second Western blot lanes are also shown in Fig 2A (left panel).
- Figure S4. Standard calibration curves of Cpd-1 and Cpd-2.
Standard calibration curves of the fluorescence of Cpd-1 (A) and Cpd-2 (B) used to determine the number of accumulated molecules shown in Fig 2B and C.
- Figure S5. Accumulation of various concentrations of Cpd-1 in RAM1292 with induction of maltose operon by 0.4% maltose.
(A) Number of Cpd-1 molecules accumulated per cell in RAM1292 grown in presence of 0.4% maltose and incubated 5 and 30 min at 37°C with various concentrations of Cpd-1 (5 to 55 µM). Bacterial pellets were lysed with glycine-HCl. The columns with bars (SDs) corresponded to measurements carried out in triplicate. (B) Standard calibration curves of Cpd-1 fluorescence used to determine the number of accumulated molecules shown in (A).
- Figure S6. Inhibition of Cpd-1 uptake by an excess of maltose in individual cells.
RAM1292 cells were grown with 0.4% maltose and pellets were resuspended extemporaneously under a DUV microscope. (A) RAM1292 cells incubated with (blue) or without (green) maltose. (B) RAM1292 cells incubated with Cpd-1 in the presence (blue) or absence (green) of maltose 10×. (C) Average curves corresponding to the frame (B). Images were acquired on a DUV microscope every 2 min and corrected using the tryptophan signal.
- Figure 3. Time-course of accumulation of Cpd-1 and Cpd-2 in RAM2808 and RAM1292 with repression or induction of the maltose operon.
(A, B) Time-course accumulation in RAM2808 cells grown without or with 0.2% arabinose (Ara, induction of the LamB expression), 0.4% maltose, and 0.2% arabinose (Malt Ara, induction of the maltose operon and LamB expression); and incubated with Cpd-1 (A) or Cpd-2 (B). The columns with bars (SDs) corresponded to measurements carried out in triplicate. (C, D) Calibration curves of the fluorescence of Cpd-1 (C) and Cpd-2 (D) used to obtain the number of molecules per cell shown in (A) and (B). (E) Time-course accumulation measured with DUV microspectrofluorimetry in RAM1292 cells grown with 0.4% glucose (Glc, repression of the maltose operon, dotted lines) or 0.4% maltose (malt, induction of the maltose operon, full lines). Cell pellets were resuspended extemporaneously under DUV microscope without (black lines) or with Cpd-1 (green lines) or Cpd-2 (purple lines). Insert: Enlargement of the Cpd-2 results.
- Figure 4. Interaction of maltotriose, maltohexaose, Cpd-1, and Cpd-2 with LamB.
(A–D) Cpd-1 interacts similarly to maltotriose with LamB. (A, B) Ion current recordings showing interaction of LamB on cis-side addition of 10 μM maltotriose (A) and 10 μM Cpd-1 (B) in 1 M KCl 10 mM Hepes, pH 7, on application of + 100 mV. (C, D) Ion current recordings showing interaction of LamB on cis-side addition of 10 μM maltotriose (C) and 10 μM Cpd-1 (D) in 1 M KCl 10 mM Hepes, pH 7, on application of −100 mV. For Cpd-2, see Fig S7. Insets show the zoomed-in view of the traces showing single interaction event. (E–H) Analysis of the ion current fluctuations reveals the kinetic parameters of the sugar interaction with LamB. (E, F) The on-rate (kon) for maltotriose or Cpd-1 (E) and for maltohexaose or Cpd-2 (F) addition at cis and trans side addition. (G, H) The residence time (τ) for maltotriose or Cpd-1 (G) and for maltohexaose or Cpd-2 (H) inside the channel after addition at cis- and trans-side addition. Note: In each experiment, we applied ± 100 Mv. As LamB is cation-selective positive voltage, it causes an ion flow from cis to trans, whereas negative voltages cause an opposite flow.
- Figure S7. Ion current recordings showing interaction of Cpd-2 with LamB.
Ion current recordings showing interaction of LamB on the cis-side addition of 2.5 μM Cpd-2 at +100 mV (left) and −100 mV (right) in 1 M KCl 10 mM Hepes, pH 7.
- Figure 5. Detection of Cpd-1 in the different cell compartments of RAM1292.
The RAM1292 cells were grown in the presence of 0.4% maltose and incubated with Cpd-1. Samples were recovered at various time points and a fractionation protocol was performed to obtain the total (TOT, periplasm + spheroplast), periplasmic (PERI), cytoplasmic (CYTO), and membrane (MB) fractions of the cells. Fluorospectrometry measurements were performed to determine the levels of Cpd-1 in each fraction. Note: Western blots were performed to confirm the distribution of specific proteins of the different cellular fractions (Fig S8A). Calibration curves were used to obtain the number of molecules per cell and per compartment (Fig S8B).
- Figure S8. Distribution of specific proteins of various cell fractions and standard calibration curves of Cpd-1.
(A) Western blot to confirm the distribution of specific proteins of the different cellular fractions of RAM1292 induced with 0.4% maltose shown in Fig 5. Antibodies were directed against EfTu (1/30,000), MalE (1/5,000) and LamB (1/10,000) that are well-known specific proteins of the cytoplasm (CYTO), periplasm (PERI), and membrane (MB) fractions respectively. (B) Standard calibration curves of Cpd-1 fluorescence used to determine the number of accumulated molecules in the different compartments of the RAM1292 cells shown in Fig 5.
- Figure 6. Behavior of Cpd-1 signal in an incubation chase study.
RAM2808 cells were grown in the absence or presence of 0.4% maltose and induced with 0.2% arabinose (Ara, induction of LamB expression; and Malt Ara, induction of the maltose operon and LamB expression). The bacterial suspension was then incubated with Cpd-1 in the absence or presence of carbonyl cyanide m-chlorophenylhydrazone (CCCP, which collapses the energy driving force of efflux pumps). (A) After incubation of the RAM2808 Malt Ara cells, samples were centrifuged, and cell pellets were resuspended in the same buffer without any Cpd-1. At 0, 15, 30, 45, 60, and 90 min, samples were recovered and analyzed. The columns with bars (standard deviations) corresponded to measurements carried out in triplicate. (B) To compare the degradation of Cpd-1 in cells according to the regulation of the maltose operon (induced or not, Malt Ara or Ara conditions, respectively), the % of signal was standardized from 0 time and the degradation level (in percent) was reported at 30 and 60 min in RAM2808 cells grown with (induction of the maltose operon) or without maltose (repression of the maltose operon) (means of three independent assays performed in triplicates). Calibration curves were used to obtain the number of molecules per cell (Fig S9).
- Figure S9. Standard calibration curves of Cpd-1.
Standard calibration curves of Cpd-1 fluorescence used to determine the number of accumulated molecules shown in Fig 6.
- Figure 7. Induction of MalE expression by Cpd-1 in RAM2808.
RAM2808 cells were grown with (top) or without (bottom) 0.4% maltose and induced with 0.2% arabinose (Ara, expression of LamB). The bacterial suspensions were incubated with Cpd-1. Samples were recovered at various time points and a cellular fractionation protocol was performed. Controls are the same bacterial suspensions incubated without Cpd-1 recovered at 30 min. Presence of MalE in the periplasmic fraction was monitored over time by Western blot analysis.
Supplementary Materials
