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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.

SUBMITTER: Yeager CM

PROVIDER: S-EPMC93320 | biostudies-literature | 2001 Dec

REPOSITORIES: biostudies-literature

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

Yeager C M CM Bottomley P J PJ Arp D J DJ

Applied and environmental microbiology 20011201 12

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 ...[more]

PMID: 11722883

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Project description:Directed evolution of toluene ortho-monooxygenase (TOM) of Burkholderia cepacia G4 previously created the hydroxylase alpha-subunit (TomA3) V106A variant (TOM-Green) with increased activity for both trichloroethylene degradation (twofold enhancement) and naphthalene oxidation (six-times-higher activity). In the present study, saturation mutagenesis was performed at position A106 with Escherichia coli TG1/pBS(Kan)TOMV106A to improve TOM activity for both chloroform degradation and naphthalene oxidation. Whole cells expressing the A106E variant had two times better naphthalene-to-1-naphthol activity than the wild-type cells (V(max) of 9.3 versus 4.5 nmol.min(-1).mg of protein(-1) and unchanged K(m)), and the regiospecificity of the A106E variant was unchanged, with 98% 1-naphthol formed, as was confirmed with high-pressure liquid chromatography. The A106E variant degrades its natural substrate toluene 63% faster than wild-type TOM does (2.12 +/- 0.07 versus 1.30 +/- 0.06 nmol.min(-1).mg of protein(-1) [mean +/- standard deviation]) at 91 microM and has a substantial decrease in regiospecificity, since o-cresol (50%), m-cresol (25%), and p-cresol (25%) are formed, in contrast to the 98% o-cresol formed by wild-type TOM. The A106E variant also has an elevated expression level compared to that of wild-type TOM, as evidenced by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Another variant, the A106F variant, has 2.8-times-better chloroform degradation activity based on gas chromatography (V(max) of 2.61 versus 0.95 nmol.min(-1).mg of protein(-1) and unchanged K(m)) and chloride release (0.034 +/- 0.002 versus 0.012 +/- 0.001 nmol.min(-1).mg of protein(-1)). The A106F variant also was expressed at levels similar to those of wild-type TOM and 62%-better toluene oxidation activity than wild-type TOM (2.11 +/- 0.3 versus 1.30 +/- 0.06 nmol.min(-1).mg of protein(-1)). A shift in regiospecificity of toluene hydroxylation was also observed for the A106F variant, with o-cresol (28%), m-cresol (18%), and p-cresol (54%) being formed. Statistical analysis was used to estimate that 292 colonies must be screened for a 99% probability that all 64 codons were sampled during saturation mutagenesis.

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

Publications

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

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