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Journal of Bacteriology, April 2000, p. 2269-2276, Vol. 182, No. 8
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

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

Zhenying Liu, Nicolas Guiliani, Corinne Appia-Ayme, Françoise Borne, Jeanine Ratouchniak, and Violaine Bonnefoy*

Laboratoire de Chimie Bactérienne, Institut de Biologie Structurale et de Microbiologie, C.N.R.S., 13402 Marseille Cedex 20, France

Received 22 November 1999/Accepted 21 January 2000

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.

* Corresponding author. Mailing address: Laboratoire de Chimie Bactérienne, Institut de Biologie Structurale et de Microbiologie, C.N.R.S., 31 chemin Joseph Aiguier, 13402 Marseille Cedex 20, France. Phone: (33) 491 164 146. Fax: (33) 491 718 914. E-mail: bonnefoy{at}ibsm.cnrs-mrs.fr.

Journal of Bacteriology, April 2000, p. 2269-2276, Vol. 182, No. 8
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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