An unusual Tn21-like transposon containing an ars operon is present in highly arsenic-resistant strains of the biomining bacterium Acidithiobacillus caldus I. Marla Tuffin, Peter de Groot, Shelly M. Deane and Douglas E. Rawlings Correspondence Douglas E. Rawlings der@sun.ac.za Department of Microbiology, Stellenbosch University, Private Bag X1, Matieland, 7602, South Africa Received 18 April 2005 Accepted 8 June 2005 A transposon, TnAtcArs, that carries a set of arsenic-resistance genes was isolated from a strain of the moderately thermophilic, sulfur-oxidizing, biomining bacterium Acidithiobacillus caldus. This strain originated from a commercial plant used for the bio-oxidation of gold-bearing arsenopyrite concentrates. Continuous selection for arsenic resistance over many years had made the bacterium resistant to high concentrations of arsenic. Sequence analysis indicated that TnAtcArs is 12 444 bp in length and has 40 bp terminal inverted repeat sequences and divergently transcribed resolvase and transposase genes that are related to the Tn21-transposon subfamily. A series of genes consisting of arsR, two tandem copies of arsA and arsD, two ORFs (7 and 8) and arsB is situated between the resolvase and transposase genes. Although some commercial strains of At. caldus contained the arsDA duplication, when transformed into Escherichia coli, the arsDA duplication was unstable and was frequently lost during cultivation or if a plasmid containing TnAtcArs was conjugated into a recipient strain. TnAtcArs conferred resistance to arsenite and arsenate upon E. coli cells. Deletion of one copy of arsDA had no noticeable effect on resistance to arsenite or arsenate in E. coli. ORFs 7 and 8 had clear sequence similarity to an NADH oxidase and a CBS-domain-containing protein, respectively, but their deletion did not affect resistance to arsenite or arsenate in E. coli. TnAtcArs was actively transposed in E. coli, but no increase in transposition frequency in the presence of arsenic was detected. Northern hybridization and reporter gene studies indicated that although ArsR regulated the 10 kb operon containing the arsenic-resistance genes in response to arsenic, ArsR had no effect on the regulation of genes associated with transposition activity. INTRODUCTION Arsenic-resistance genes are widely distributed in Bacteria, Archaea and also in some Eukarya, and have been extensively studied. Although the gene order and number of ars genes varies, two of the most commonly encountered sets of ars genes are the arsRBC genes such as found on the chro- mosome of Escherichia coli (Carlin et al., 1995) and the arsRDABC genes such as found on plasmid R773 (Chen et al., 1986). ArsR is a negative regulator of the ars operon (Wu & Rosen, 1991), ArsD is a second repressor (Wu & Rosen, 1993) that prevents the ars operon from being over- expressed, ArsA is an ATPase that associates with ArsB and links arsenite export to ATP hydrolysis (Dey et al., 1994), ArsB is a membrane-associated arsenite export pump (Tisa & Rosen, 1989) and ArsC is an arsenate reductase (Ji & Silver, 1992) that converts arsenate [As(V)] to arsenite [AS(III)], which can then be pumped out through the action of ArsB. Other genes that have been reported to be asso- ciated with arsenic resistance are arsH, the product of which has an unknown function and which has been reported to be required for arsenic resistance in the case of a Yersinia enterolitica pYV plasmid-located ars operon (Neyt et al., 1997) but not in other cases where it has been found (Butcher et al., 2000), and arsM, a putative arsenite- methyltransferase (Wang et al., 2004). Continuous-flow, stirred-tank based processes for the bio- oxidation of gold-bearing arsenopyrite concentrates were developed during the 1980s (Rawlings & Silver, 1995). A number of large-scale industrial plants that use the above process as a pre-treatment step for the subsequent recovery of gold by cyanidation have been built in several countries (Dew et al., 1997). Continuous-flow tanks that are used for the bio-oxidation of arsenopyrite concentrates and that operate at 40 u C are dominated by a mixture of the sulphur-oxidizing bacterium Acidithiobacillus caldus and the iron-oxidizing bacterium Leptospirillum ferriphilum (Rawlings et al., 1999). When arsenopyrite concentrates The GenBank/EMBL/DDBJ accession number for the nucleotide sequence of TnAtcArs is AY821803. 0002-8131 G 2005 SGM Printed in Great Britain 3027 Microbiology (2005), 151, 3027–3039 DOI 10.1099/mic.0.28131-0