Mutagenesis by reverse genetics of the acidophilic chemolithoautotrophic Acidithiobacillus ferrooxidans: construction of a recA mutant

摘要

The study of mutants in which a protein of interest is no longer synthesized can help to establish the function of this protein. In the case of the acidophilic chemolithoautotrophic Acidithiobacillus ferrooxidans, the absence of genetic tools has impaired the understanding of its physiology. As a first approach to At. ferrooxidans genetics, we have tested marker exchange mutagenesis with the construction of a recA mutant. A mobilizable suicide plasmid carrying the ATCC33020 recA gene disrupted by a kanamycin resistant gene was transferred from Escherichia coli to At. ferrooxidans ATCC33020 by conjugation. PCR, Southern hybridization and sequence analyses showed that the stable kanamycin resistant clones have the kanamycin resistance gene inserted within the recAgene indicating that the mutated allele was transferred from the suicide plasmid to the chromosome by homologous recombination. These mutants exhibited an increased sensitivity to UV and γ irradiation confirming the disruption of the recA gene. The successful construction of a knock-out recAmutant demonstrates the feasibility of reverse genetics in At. ferrooxidans ATCC33020 strain by using conjugation as a genetic transfer technique.