Construction and Characterization of a recA Mutant of Thiobacillus ferrooxidans by Marker Exchange Mutagenesis

To constructThiobacillus 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) fromEscherichia coli to two privateT. ferrooxidans strains (BRGM1 and Tf-49) and to two collection strains (ATCC 33020 and ATCC 19859) by conjugation was analyzed. To knock out theT. ferrooxidans recA gene, a mobilizable suicide plasmid carrying the ATCC 33020recA gene disrupted by a kanamycin resistance gene was transferred fromE. coli toT. 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 therecA gene, indicating that therec A::Ω-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 γ irradiation compared to the wild-type strain. However, theT. ferrooxidans recA mutants are less sensitive to these physical DNA-damaging agents than therecA mutants described in other bacterial species, suggesting that RecA plays a minor role in DNA repair inT. ferrooxidans .