Requirement of DNA repair mechanisms for survival of Burkholderia cepacia G4 upon degradation of trichloroethylene

Abstract

A Tn5-based mutagenesis strategy was used to generate a collection of trichloroethylene (TCE)-sensitive (TCS) mutants in order to identify repair systems or protective mechanisms that shield Burkholderia cepacia G4 from the toxic effects associated with TCE oxidation. Single Tn5 insertion sites were mapped within open reading frames putatively encoding enzymes involved in DNA repair (UvrB, RuvB, RecA, and RecG) in 7 of the 11 TCS strains obtained (4 of the TCS strains had a single Tn5 insertion within a uvrB homolog). The data revealed that the uvrB-disrupted strains were exceptionally susceptible to killing by TCE oxidation, followed by the recA strain, while the ruvB and recG strains were just slightly more sensitive to TCE than the wild type. The uvrB and recA strains were also extremely sensitive to UV light and, to a lesser extent, to exposure to mitomycin C and H(2)O(2). The data from this study establishes that there is a link between DNA repair and the ability of B. cepacia G4 cells to survive following TCE transformation. A possible role for nucleotide excision repair and recombination repair activities in TCE-damaged cells is discussed.

FIG. 1

Colony growth of wild-type B. cepacia G4 (WT G4) and select TCS mutants on minimal medium agar plates with toluene vapors with and without TCE vapors. Colonies were spotted in duplicate on each plate.

FIG. 2

Schematic maps of the DNA fragments self-cloned from the TCS mutants. The open triangles indicate the locations of the Tn5-OT182 insertions in the TCS mutants. The recA, ruvB, uvrB, and recG homologs are indicated by thick lines, and the directions of transcription are indicated by arrowheads. The nucleotide sequence of the recA upstream region is shown in the inset, and the putative transcriptional start site (+1) and −35 and −10 promoter sequences are underlined. The putative LexA binding region (SOS box) is enclosed in a box.

FIG. 3

Time courses for recovery of growth by TCE-treated cells of wild-type B. cepacia G4 (▪), TCS-1 (uvrB) (●), TCS-4 (uvrB) (▴), TCS-8 (ruvB) (♦), TCS-12 (recA) (□), and TCS-14 (recG) (○). Cells were grown overnight with toluene vapors and incubated with (A) or without (B) TCE for 30 min. Portions of each cell suspension were then added to vials containing minimal medium with 20 mM sodium lactate, and culture growth (at 30°C with shaking) was monitored by determining OD₆₀₀.

FIG. 4

Survival of wild-type B. cepacia G4 (▪), TCS-1 (uvrB) (●), TCS-8 (ruvB) (♦), TCS-12 (recA) (□), and TCS-14 (recG) (○) following exposure to TCE (A), UV light (B), mitomycin C (C), or H₂O₂ (D). Toluene-grown cells were exposed to each agent as described in Materials and Methods, diluted in phosphate buffer, and spread on R2A agar plates. After 3 days of incubation at 30°C, the number of colonies per plate was determined and compared to the number of colonies on control plates containing untreated cells to determine the surviving fraction.