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.Trichloroethylene (TCE), a suspected human carcinogen (17), has been used extensively as a metal degreaser, fumigant, and solvent for dry cleaning and in other commercial applications. Because of its widespread use and persistence, TCE is one the most commonly detected organic pollutants at hazardous waste sites and in municipal groundwater supplies in the United States (24, 48). Although it has not been demonstrated that microorganisms can utilize TCE as a growth-supporting substrate under aerobic conditions, a number of bacteria are able to degrade TCE cometabolically; in this process nonspecific oxygenases catalyze the initial transformation (1,8,44).The practicality of utilizing bacteria to degrade TCE via aerobic cometabolism has been questioned, however, due to the cytotoxicity that is almost universally associated with this process. Loss of TCE-degradative activity is often observed with whole cells during TCE transformation (34,36,43,45,49), and each of the TCE-degrading enzymes that have been purified to homogeneity and examined to date (toluene dioxygenase, toluene 2-monooxygenase, and soluble methane monooxygenase) exhibits turnover-dependent inactivation upon TCE oxidation (12,22,33). Additionally, TCE degradation can result in injuries that adversely affect more basic cellular functions, such as general respiratory activity and cell viability (4,16,43,49). Although the exact nature of the destructive species remains unknown, it has been proposed that acyl chlorides, generated from hydrolysis or rearrangement of TCE epoxide (monooxygenase-catalyzed reactions) or TCEdioxetane (dioxygenase-catalyzed reactions), cause damage by alkylating various cellular constituents (12,22,33,43,46). Since cellular toxicity can potentially limit the sustainability of TCE biodegradation under aerobic conditions, a concerted effort has been directed towards identifying strains t...

The Extracytoplasmic Function Sigma Factor σ S Protects against both Intracellular and Extracytoplasmic Stresses in Staphylococcus aureus

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Acinetobacter baumannii RecA Protein in Repair of DNA Damage, Antimicrobial Resistance, General Stress Response, and Virulence

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