Modulation of glucose transport causes preferential utilization of aromatic compounds in Pseudomonas putida CSV86

Abstract

Pseudomonas putida CSV86 utilizes aromatic compounds in preference to glucose and coutilizes aromatics and organic acids. Protein analysis of cells grown on different carbon sources, either alone or in combination, revealed that a 43-kDa periplasmic-space protein was induced by glucose and repressed by aromatics and succinate. Two-dimensional gel electrophoresis and liquid chromatography-tandem mass spectrometry analysis identified this protein as closely resembling the sugar ABC transporter of Pseudomonas putida KT2440. A partially purified 43-kDa protein showed glucose binding activity and was specific for glucose. The results demonstrate that the aromatic- and organic acid-mediated repression of a periplasmic-space glucose binding protein and consequent inhibition of glucose transport are responsible for this strain's ability to utilize aromatics and organic acids in preference to glucose.

FIG. 1.

Growth profiles of Pseudomonas putida CSV86 on different carbon sources. The growth profiles of CSV86 (▿) on naphthalene (0.1%) plus glucose (0.25%) (A), benzyl alcohol (0.1%) plus glucose (0.25%) (B), and succinate (0.25%) plus glucose (0.25%) (C) are shown. The growth profile for a single carbon source, such as naphthalene (0.1%), benzyl alcohol (0.1%), or succinate (0.25%), is represented by circles. The decreasing levels of glucose in the spent medium for panels A, B, and C are shown by inverted triangles.

FIG. 2.

[¹⁴C]glucose uptake by whole cells of Pseudomonas putida CSV86. Cells were grown on naphthalene for 8 h (○), glucose for 22 h (⋄), and naphthalene plus glucose for 4 h (▿), 8 h (□), or 22 h (▵) (A); benzyl alcohol for 8 h (○), glucose for 22 h (⋄), and benzyl alcohol plus glucose for 4 h (▿), 8 h (□), or 22 h (▵) (B); and succinate for 7 h (○), glucose for 22 h (⋄), and succinate plus glucose for 7 h (▿) and 22 h (□) (C).

FIG. 3.

Growth profiles and glucose binding activities of the periplasmic-space protein from glucose-grown cells of Pseudomonas putida CSV86 spiked with the second carbon source. (A) Growth profile of CSV86 precultured on either glucose (○), naphthalene (□), or succinate (▵) and then transferred onto glucose (0.25%) as the sole carbon source. (B) Growth profile of glucose-grown CSV86 spiked (indicated by an arrow) at 15 h (mid-log-phase culture) with either naphthalene (•; 0.1%) or succinate (★; 0.25%). (C and D) [¹⁴C]glucose binding activity and SDS-PAGE profile (inset) of the periplasmic-space protein of glucose-grown cells spiked with naphthalene (C) or succinate (D). The [¹⁴C]glucose binding activity of 15-h glucose-grown cells was taken as 100%. The probable glucose-binding protein is indicated by an arrow. Lane M, standard molecular mass marker protein (43 kDa).

FIG. 4.

(A to D) SDS-PAGE (10%) analysis of periplasmic-space proteins from Pseudomonas putida CSV86. Cells were grown on glucose (Glc), naphthalene (Nap), benzyl alcohol (Balc), or succinate (Suc) as well as on dual carbon sources for the durations indicated. Each lane contains 15 μg protein. Lane M contains standard molecular mass markers. Thick arrowheads denote the probable glucose binding periplasmic protein (43 kDa). Thin arrows represent significant changes observed in the levels of other proteins. (E to H) [¹⁴C]glucose binding activity of the periplasmic-space protein fractions obtained from CSV86 cells grown on either naphthalene, benzyl alcohol, or succinate are represented by black bars (F to H, respectively), while binding activity from cells grown on glucose, naphthalene plus glucose, benzyl alcohol plus glucose, or succinate plus glucose are represented by gray bars (E to H, respectively). Growth profiles for respective double carbon sources are shown (▿).

FIG. 5.

2-D gel electrophoretic analysis of the periplasmic-space protein fraction of Pseudomonas putida CSV86. Cells were grown on naphthalene (0.1%) (A), on naphthalene (0.1%) plus glucose (0.25%) for 7 h (B) and 15 h (C), and on glucose (0.25%) (D). Spots marked with circles were excised after visual comparison of profiles and analyzed further by LC-MS/MS as described in Materials and Methods. IEF, isoelectric focusing.

FIG. 6.

Partial purification and substrate specificity of the periplasmic-space glucose binding protein from Pseudomonas putida CSV86. (A) Gel filtration protein elution profile (Sephacryl S-200HR) of periplasmic-space proteins (○) from glucose-grown cells of CSV86 and their [¹⁴C]glucose binding activities (•). The gel filtration column was calibrated by using β-amylase (200 kDa), alcohol dehydrogenase (150 kDa), bovine serum albumin (66 kDa), and carbonic anhydrase (29 kDa). Inset: SDS-PAGE analysis (with Coomassie staining) of the fractions showing the highest [¹⁴C]glucose binding activities: lane 1, fraction 39 (maximum [¹⁴C]glucose binding activity); lane 2, fraction 40; lane 3, fraction 41; and lane 4, molecular mass marker for Ovalbumin (43 kDa). The arrow shows the 43-kDa protein. (B) Specificity of the 43-kDa glucose binding protein. See Materials and Methods for details.