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. 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.
Liu
Z
Z
Laboratoire de Chimie Bactérienne, Institut de Biologie Structurale et de Microbiologie, C.N.R.S., 13402 Marseille Cedex 20, France.
Guiliani
N
N
Appia-Ayme
C
C
Borne
F
F
Ratouchniak
J
J
Bonnefoy
V
V
D003227:Conjugation, Genetic; D004926:Escherichia coli / Q000235:genetics; D005720:Gamma Rays; D005819:Genetic Markers; D016296:Mutagenesis; D010957:Plasmids; D011836:Radiation Tolerance; D011938:Rec A Recombinases / Q000235:genetics*; D013855:Thiobacillus / Q000235:genetics* / Q000528:radiation effects; D014466:Ultraviolet Rays
D005819:Genetic Markers; D011938:Rec A Recombinases