A chromosomally based tod-luxCDABE whole-cell reporter for benzene, toluene, ethybenzene, and xylene (BTEX) sensing

Abstract

A tod-luxCDABE fusion was constructed and introduced into the chromosome of Pseudomonas putida F1, yielding the strain TVA8. This strain was used to examine the induction of the tod operon when exposed to benzene, toluene, ethylbenzene, and xylene (BTEX) compounds and aqueous solutions of JP-4 jet fuel constituents. Since this system contained the complete lux cassette (luxCDABE), bacterial bioluminescence in response to putative chemical inducers of the tod operon was measured on-line in whole cells without added aldehyde substrate. There was an increasing response to toluene concentrations from 30 micrograms/liter to 50 mg/liter, which began to saturate at higher concentrations. The detection limit was 30 micrograms/liter. There was a significant light response to benzene, m- and p-xylenes, phenol, and water-soluble JP-4 jet fuel components, but there was no bioluminescence response upon exposure to o-xylene. The transposon insertion was stable and had no negative effect on cell growth.

FIG. 1

(A) Construction of mini-Tn5Kmtod-lux. A/X and Nh/X represent the AvrII-XbaI and NheI-XbaI heterologous cloning sites, respectively. N, NotI; Sa, SalI; X, XbaI. (B) Cloning plasmid pLJS with unique restriction sites. A, AvrII; Ac, AccI; Ap, ApaI; B, BamHI; Bs, BstXI; C, ClaI; D, DraII; E, EcoRI; Ea, EagI; EV, EcoRV; H, HindIII; Hc, HincII; K, KpnI; Nh, NheI; P, PstI; S, SpeI; Sc, SacI; ScII, SacII; Sm, SmaI; Xh, XhoI.

FIG. 2

Bioluminescence and growth of TVA8 and growth of F1 on toluene vapor under batch conditions. ○, □, and ▵ represent individual replicates of bioluminescence readings over time. The solid squares (TVA8) and circles (F1) represent the average OD₅₄₆ of three replicates.

FIG. 3

Specific bioluminescence of TVA8 grown on toluene vapor. The regression line equation is y = 116x − 418 (r² = 0.982). (The first four time points were not included in the linear regression because the organisms were in lag phase.)

FIG. 4

Bioluminescence response of TVA8 to increasing concentrations of toluene after a 2-h exposure. Values are averages of three replicates and have been normalized to the cell density (OD₅₄₆).