Cloning and characterization of the chromosomal arsenic resistance genes from Acidithiobacillus caldus and enhanced arsenic resistance on conjugal transfer of ars genes located on transposon TnAtcArs

Department of Microbiology, University of Stellenbosch, Private Bag X1, Matieland 7602, South Africa Correspondence Douglas E. Rawlings der{at}sun.ac.za All strains of the moderately thermophilic, acidophilic, sulphur-oxidizing bacterium Acidithiobacillus caldus that have been tested contain a set of chromosomal arsenic resistance genes. Highly arsenic-resistant strains isolated from commercial arsenopyrite bio-oxidation tanks contain additional transposon-located (Tn AtcArs ) arsenic resistance genes. The chromosomal At. caldus ars genes were cloned and found to consist of arsR and arsC genes transcribed in one direction, and arsB in the opposite direction. The arsRC genes were co-transcribed with ORF1, and arsB with ORF5 in both At. caldus and Escherichia coli , although deletion of ORFs 1 and 5 did not appear to affect resistance to arsenate or arsenite in E. coli . ORFs 1 and 5 have not previously been reported as part of the ars operons, and had high amino acid identity to hypothetical proteins from Polaromonas naphthalenivorus (76 %) and Legionella pneumophila (60 %), respectively. Reporter-gene studies showed that the arsenic operon of transposon origin (Tn AtcArs ) was expressed at a higher level, and was less tightly regulated in E. coli than were the At. caldus ars genes of chromosomal origin. Plasmid pSa-mediated conjugal transfer of Tn AtcArs from E. coli to At. caldus strains lacking the transposon was successful, and resulted in greatly increased levels of resistance to arsenite. Abbreviations: NCBI, National Centre for Biotechnology Information The GenBank/EMBL/DDBJ accession number for the sequence of the insert of pAtcars4 reported in this paper is DQ810790.