Toluene-degrading bacteria are chemotactic towards the environmental pollutants benzene, toluene, and trichloroethylene

Abstract

The bioremediation of polluted groundwater and toxic waste sites requires that bacteria come into close physical contact with pollutants. This can be accomplished by chemotaxis. Five motile strains of bacteria that use five different pathways to degrade toluene were tested for their ability to detect and swim towards this pollutant. Three of the five strains (Pseudomonas putida F1, Ralstonia pickettii PKO1, and Burkholderia cepacia G4) were attracted to toluene. In each case, the response was dependent on induction by growth with toluene. Pseudomonas mendocina KR1 and P. putida PaW15 did not show a convincing response. The chemotactic responses of P. putida F1 to a variety of toxic aromatic hydrocarbons and chlorinated aliphatic compounds were examined. Compounds that are growth substrates for P. putida F1, including benzene and ethylbenzene, were chemoattractants. P. putida F1 was also attracted to trichloroethylene (TCE), which is not a growth substrate but is dechlorinated and detoxified by P. putida F1. Mutant strains of P. putida F1 that do not oxidize toluene were attracted to toluene, indicating that toluene itself and not a metabolite was the compound detected. The two-component response regulator pair TodS and TodT, which control expression of the toluene degradation genes in P. putida F1, were required for the response. This demonstration that soil bacteria can sense and swim towards the toxic compounds toluene, benzene, TCE, and related chemicals suggests that the introduction of chemotactic bacteria into selected polluted sites may accelerate bioremediation processes.

FIG. 1

Initial reactions in the five bacterial pathways for aerobic degradation of toluene in strains P. putida F1, P. putida PaW15 (a leucine auxotroph of strain mt-2 [41]), B. cepacia G4, R. pickettii PKO1, and P. mendocina KR1. P. putida F1 utilizes a dioxygenase-initiated pathway for toluene degradation. G4, PKO1, and KR1 initiate toluene degradation with toluene 2-, 3-, and 4-monooxygenases, respectively. PaW15 carries the TOL plasmid and oxidizes the methyl group of toluene.

FIG. 2

Chemotactic responses of the five bacterial strains (see the legend to Fig. 1) to toluene in agarose plug assays 5 min after addition of cells. Cells were grown in the presence of toluene as described in Materials and Methods.

FIG. 3

(A) Time course of the chemotactic response of P. putida F1 to toluene in modified capillary assays at an ×40 magnification. P. putida F1 was grown in the presence of toluene as described in Materials and Methods. Capillaries contained 1.4 mM toluene. No response was seen when capillaries contained only agarose and chemotaxis buffer. (B) Chemotactic responses of P. putida F1 to benzene, ethylbenzene, propylbenzene, TFT, TCE, DCE, and PCE after growth in the presence of toluene. Plug assays were carried out as described in Materials and Methods. Cells were incubated for 5 min in the presence of the agarose plugs. Images were cropped.

FIG. 4

(A) Pathway for the degradation of toluene in P. putida F1. Gene products catalyzing each step are indicated in panel B. (B) Genetic organization of the toluene degradation gene cluster and surrounding catabolic genes in P. putida F1. Solid arrows indicate repeated sequences (4). Also shown are the functions of the tod gene products and the locations of mutations of interest. Triangles indicate the insertion of a kanamycin cassette, and asterisks indicate spontaneous mutations. (C) Chemotactic responses of P. putida F1 and mutant strains after 5 min in the presence of toluene-containing agarose plugs. Strains were grown with pyruvate and toluene. Plug assays were performed as described in Materials and Methods; images were cropped. Cells grown with pyruvate alone did not respond (data not shown). No response was observed when toluene was omitted from the plug (data not shown). The response of each strain was tested in the modified capillary assay (data not shown), and results were consistent with the plug assay results.