Utilization of trihalogenated propanes by Agrobacterium radiobacter AD1 through heterologous expression of the haloalkane dehalogenase from Rhodococcus sp. strain M15-3

Abstract

Trihalogenated propanes are toxic and recalcitrant organic compounds. Attempts to obtain pure bacterial cultures able to use these compounds as sole carbon and energy sources were unsuccessful. Both the haloalkane dehalogenase from Xanthobacter autotrophicus GJ10 (DhlA) and that from Rhodococcus sp. strain m15-3 (DhaA) were found to dehalogenate trihalopropanes to 2,3-dihalogenated propanols, but the kinetic properties of the latter enzyme are much better. Broad-host-range dehalogenase expression plasmids, based on RSF1010 derivatives, were constructed with the haloalkane dehalogenase from Rhodococcus sp. strain m15-3 under the control of the heterologous promoters P(lac), P(dhlA), and P(trc). The resulting plasmids yielded functional expression in several gram-negative bacteria. A catabolic pathway for trihalopropanes was designed by introducing these broad-host-range dehalogenase expression plasmids into Agrobacterium radiobacter AD1, which has the ability to utilize dihalogenated propanols for growth. The recombinant strain AD1(pTB3), expressing the haloalkane dehalogenase gene under the control of the dhlA promoter, was able to utilize both 1,2,3-tribromopropane and 1,2-dibromo-3-chloropropane as sole carbon sources. Moreover, increased expression of the haloalkane dehalogenase resulted in elevated resistance to trihalopropanes.

FIG. 1

Schematic organization of the promoter and dehalogenase coding region of the constructed expression vectors. Rhodococcus DNA is shown as a hatched block. Thin arrows indicate the direction of transcription of the dhaA gene. Thick arrows represent the promoters. The shaded blocks indicate the transcriptional terminator. Only relevant restriction sites are shown.

FIG. 2

Effect of increasing concentrations of trihalogenated propanes on growth of A. radiobacter strains AD1 (●), AD1(pTB1) (▴), and AD1(pTB3) (■) growing on 5 mM 1,3-dichloro-2-propanol. The turbidity (OD₄₅₀) of the cultures was measured after 7 days of cultivation at 30°C. Initial OD₄₅₀ values ranged from 0.5 to 0.7. (A) 1,2,3-tribromopropane; (B) 1,2-dibromo-3-chloropropane; (C) 1,2,3-trichloropropane.

FIG. 3

Growth and degradation of trihalogenated propanes by A. radiobacter AD1(pTB3). Cultures were grown aerobically at room temperature in MMY medium supplemented with 1 mM 1,2,3-tribromopropane (4 pulses), 1.2 mM 1,2-dibromo-3-chloropropane (2 pulses), or 1.6 mM 1,2,3-trichloropropane as carbon source. (A) 1,2,3-Tribromopropane (○), 2,3-dibromo-1-propanol (●), OD₄₅₀ (▴); (B) 1,2-dibromo-3-chloropropane (○), 2-bromo-3-chloro-1-propanol (●), OD₄₅₀ (▴); (C) 1,2,3-trichloropropane (○), 2,3-dichloro-1-propanol (●), OD₄₅₀ (▴); (D) (sterile controls) 1,2,3-tribromopropane (●), 1,2-dibromo-3-chloropropane (▿), 1,2,3-trichloropropane (■).

FIG. 4

Proposed pathway for complete degradation of trihalogenated propanes by A. radiobacter AD1(pTB3). X, chloride or bromide.