Identification and characterization of four strains of Acidithiobacillus ferrooxidans isolated from different sites in China

SummaryFour strains of Acidithiobacillus ferrooxidans (A. ferrooxidans), AF1, AF2, AF3 and AFc, were isolated from samples with different geological sources using a 9K medium. These four isolates were identified as A. ferrooxidans by phenotypic and 16S rDNA sequence analyses. All four isolates were able to use ferrous ion (Fe2+), elemental sulfur (S0) or pyrite as a sole energy source, but they showed differences in pH optima and range of activity, optimum temperature of activity, resistance to chloride (KCl) and heavy metal ions, and oxidation rates of Fe2+, S0 and pyrite. AF3 was the most active strain when using Fe2+ as the energy source, while AFc grew best using pyrite as the energy source. AF2 appeared to differ from the other three strains in substrate utilization, as it oxidizes S0 and pyrite more effectively than Fe2+. RAPD analysis of genomic DNA from these isolates showed that banding profiles of their genomic DNA exhibited some differences, and the genomic banding profile of AF2 was significantly different from that of others. To obtain an insight into the molecular biology of the process of the energy production of these strains, several genes involved in the iron respiratory chain were cloned and sequenced, including Fe2+ oxidase (iro), rusticyanin (rus) and subunit III of aa3-type cytochrome oxidase (cox C) genes. The results revealed that the iro gene can be cloned from all of the four strains and the nucleotide sequences were shown to be completely identical in each. However, rus and coxC genes could be amplified only from AF1, AF3 and AFc, not from AF2. These results suggested that the phenotypic differences of the four strains of A. ferrooxidans from different sites correlated with their genetic polymorphism, which may result from the different environments in which they lived, and that the strain AF2 was phenotypically and genetically significantly different from the other three strains.