Construction and characterization of a recA mutant of Thiobacillus ferrooxidans by marker exchange mutagenesis

To construct Thiobacillus ferrooxidans mutants by marker exchange mutagenesis, a genetic transfer system is required. The transfer of broad-host-range plasmids belonging to the incompatibility groups IncQ (pKT240 and pJRD215), IncP (pJB3Km1), and IncW (pUFR034) from Escherichia coli to two private T...

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Bibliographic Details
Published in:Journal of bacteriology 2000-04, Vol.182 (8), p.2269-2276
Main Authors: Liu, Z, Guiliani, N, Appia-Ayme, C, Borne, F, Ratouchniak, J, Bonnefoy, V
Format: Article
Language:English
Subjects:
Bacteriology
Conjugation, Genetic
Escherichia coli
Escherichia coli - genetics
Gamma Rays
Genetic Markers
Genetics
Genetics and Molecular Biology
kanamycin
Mutagenesis
Mutation
plasmid pJB3Km1
plasmid pJRD215
plasmid pKT240
plasmid pUFR034
Plasmids
Radiation Tolerance
Rec A Recombinases - genetics
recA gene
Thiobacillus - genetics
Thiobacillus - radiation effects
Thiobacillus ferrooxidans
Ultraviolet Rays
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Summary:To construct Thiobacillus ferrooxidans mutants by marker exchange mutagenesis, a genetic transfer system is required. The transfer of broad-host-range plasmids belonging to the incompatibility groups IncQ (pKT240 and pJRD215), IncP (pJB3Km1), and IncW (pUFR034) from Escherichia coli to two private T. ferrooxidans strains (BRGM1 and Tf-49) and to two collection strains (ATCC 33020 and ATCC 19859) by conjugation was analyzed. To knock out the T. ferrooxidans recA gene, a mobilizable suicide plasmid carrying the ATCC 33020 recA gene disrupted by a kanamycin resistance gene was transferred from E. coli to T. ferrooxidans ATCC 33020 by conjugation under the best conditions determined. The two kanamycin-resistant clones, which have retained the kanamycin-resistant phenotype after growth for several generations in nonselective medium, were shown to have the kanamycin resistance gene inserted within the recA gene, indicating that the recA::Omega-Km mutated allele was transferred from the suicide plasmid to the chromosome by homologous recombination. These mutants exhibited a slightly reduced growth rate and an increased sensitivity to UV and gamma irradiation compared to the wild-type strain. However, the T. ferrooxidans recA mutants are less sensitive to these physical DNA-damaging agents than the recA mutants described in other bacterial species, suggesting that RecA plays a minor role in DNA repair in T. ferrooxidans.
ISSN:0021-9193
1098-5530
DOI:10.1128/JB.182.8.2269-2276.2000