A transposon-located arsenic resistance mechanism from a strain of Acidithiobacillus caldus isolated from commercial, arsenopyrite biooxidation tanks

The moderately thermophilic, sulfur-oxidizing bacterium Acidithiobacillus caldus is an important role player in continuous-flow biooxidation processes associated with biomining. Biooxidation of arsenopyrite concentrates by a combination of Leptospirillum and At. caldus results in the release of soluble arsenic into the surrounding environment. Arsenate and arsenite are toxic to most organisms, yet At. caldus is able to thrive in this harsh environment. The purpose of this study was to identify and investigate arsenic resistance genes in At. caldus and compare its ars operon with those previously studied in other bacteria. A gene bank of a South African At. caldus strain #6 was constructed and transformed into an Eseherichia coli arsenic-sensitive mutant strain to identify clones containing At. caldus arsenic resistance genes. A clone with an insert of 6.4 kb was selected and after two cycles of chromosome-walking a further 5.6 kb of DNA upstream of the original clone was isolated. This region was sequenced and found to have an unusual ars operon with a functional arsB gene, two arsD-like and two arsA-like genes. The arsenic resistance mechanism appears to be located on a Tn21-like transposon and includes transposase and resolvase-like genes with the entire assemblage being flanked by Tn21-like 38 bp inverted repeats (IR). Southern hybridization experiments using the arsB gene as a probe against total DNA from six different At. caldus strains indicated that the three South African strains which had been previously exposed to arsenic gave a positive hybridization signal, while three other At. caldus strains (KU, BC13 and C-SH12) did not. Southern hybridization of pulse field gels indicated that the arsB gene was chromosomally encoded. Genes encoding for NADH oxidase-like and IMP dehydrogenase-like enzymes were situated between the arsA and arsB genes. PCR and Southern hybridization experiments revealed that this unusual arrangement was not an artefact of cloning but that the 2.1 kb NADH oxidase-like and IMP dehydrogenase-like gene containing region was present between the arsA and arsB genes in all three South African strains. When transformed into the E. coli arsenic-sensitive mutant, the transposon-located system conferred increased resistance to arsenite, but not to arsenate. This indicated that the NADH oxidase-like gene did not function as an arsenate reductase in E. coli. It is possible that increased exposure to arsenic placed selection pressure on the three South African strains to acquire a Tn21-like transposon containing an ars operon to enhance their arsenic resistance. (C) 2003 Elsevier B.V. All rights reserved.