Role of Pseudomonas putida tol-oprL gene products in uptake of solutes through the cytoplasmic membrane

Abstract

Proteins of the Tol-Pal (Tol-OprL) system play a key role in the maintenance of outer membrane integrity and cell morphology in gram-negative bacteria. Here we describe an additional role for this system in the transport of various carbon sources across the cytoplasmic membrane. Growth of Pseudomonas putida tol-oprL mutant strains in minimal medium with glycerol, fructose, or arginine was impaired, and the growth rate with succinate, proline, or sucrose as the carbon source was lower than the growth rate of the parental strain. Assays with radiolabeled substrates revealed that the rates of uptake of these compounds by mutant cells were lower than the rates of uptake by the wild-type strain. The pattern and amount of outer membrane protein in the P. putida tol-oprL mutants were not changed, suggesting that the transport defect was not in the outer membrane. Consistently, the uptake of radiolabeled glucose and glycerol in spheroplasts was defective in the P. putida tol-oprL mutant strains, suggesting that there was a defect at the cytoplasmic membrane level. Generation of a proton motive force appeared to be unaffected in these mutants. To rule out the possibility that the uptake defect was due to a lack of specific transporter proteins, the PutP symporter was overproduced, but this overproduction did not enhance proline uptake in the tol-oprL mutants. These results suggest that the Tol-OprL system is necessary for appropriate functioning of certain uptake systems at the level of the cytoplasmic membrane.

FIG. 1.

Physical and transcriptional organization of the tol-oprL cluster of P. putida KT2440. The solid arrows represent the different tol genes, their relative sizes, and the directions of transcription. The positions of the P₁ and P_L promoters are indicated (33).

FIG. 2.

Strategy used to construct an oprB mutant of P. putida. (A) Physical map of pCHESIΩKm. Single restriction sites are indicated by boldface type. (B) Physical map of pCHESI-B and cointegration into the host chromosome to obtain an oprB mutant. Details of the cloning and cointegration procedures are described in the text.

FIG. 3.

Uptake of different carbon sources by P. putida KT2440 and its isogenic mutants. Uptake of glucose (A and B) and glycerol transport (C and D) by whole cells (A and C) or spheroplasts (B and D) of the P. putida wild-type strain (•) and P. putida QX (tolQ mutant) (⋄), BX (tolB mutant) (▴), and oprB (□) mutant strains. Cells were grown in minimal glucose medium until the exponential growth phase was reached, and uptake was analyzed as described in Materials and Methods.

FIG. 4.

Uptake of proline by whole cells of different strains of P. putida. The strains used were P. putida KT2440 (squares), P. putida CRR216 (putP mutant) (circles), P. putida BX (tolB mutant) (triangles), P. putida QX (tolQ mutant) (diamonds), and P. putida QΩ (tolQ mutant) (rectangles) bearing pBBR1MCS-5 as a negative control (open symbols) or pBBRPutP (solid symbols). Cells were grown in LB medium until an OD₆₆₀ of ∼0.5 was reached, washed once, resuspended in M9 minimal medium, and incubated for 3 h. Uptake was measured as described in Materials and Methods.

FIG. 5.

Determination of Δψ in P. putida KT2440 (WT) and tol::xylE mutant strains (P. putida QX, RX, AX, BX, and DOT-OX2). Cells were grown in LB medium, harvested in the exponential phase of growth (OD₆₆₀, ∼0.4), and converted to spheroplasts as described in Materials and Methods. Generation of the proton motive force by spheroplasts was monitored by monitoring the fluorescence quenching of DiSC₂(5) as described in Materials and Methods. The arrows indicate the times when the fluorescence probe and valinomycin (V) were added.