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

Department of Microbiology, Stellenbosch University, Private Bag X1, Matieland, 7602, South Africa

Correspondence
Douglas E. Rawlings
der{at}sun.ac.za


   ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
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.


The GenBank/EMBL/DDBJ accession number for the nucleotide sequence of TnAtcArs is AY821803.


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
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 chromosome 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 overexpressed, 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 associated 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 °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 are bio-oxidized, levels of arsenic frequently reach saturation, and soluble arsenate concentrations of 12 g l–1 and arsenite concentrations of 3–6 g l–1 have been reported. One of the difficulties encountered in the initial stages of the process development was that the micro-organisms responsible for mineral bio-oxidation were sensitive to the high concentrations of arsenic released during mineral oxidation. The operation of a continuous-flow tank aeration system over a period of several years, where fast-growing arsenopyrite-bio-oxidizing cells displace the more readily washed out slow-growing cells, resulted in the selection of highly arsenic-resistant microbes (Rawlings & Silver, 1995).

We obtained cultures of At. caldus strains that had been exposed to selection for high levels of arsenic resistance in industrial tanks and other strains of the same species that had not been similarly selected. By investigating the arsenic-resistance genes from these bacteria we wished to discover how the arsenic-resistant strains had become resistant to such high levels of arsenic. We had previously reported the cloning of pieces of an arsenic-resistance operon that appeared to be located on a Tn21-like transposon (de Groot et al., 2003; Tuffin et al., 2004). Here we report on the isolation and sequence of the intact transposon that is present only in highly arsenic-resistant strains, the structure of the ars operon containing several previously unidentified genes, and the ability of this system to function in E. coli.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Media, bacterial strains and plasmids.
Bacterial strains and plasmids used in this study are shown in Table 1. E. coli strains were grown in Luria–Bertani (LB) broth medium (Sambrook et al., 1989) with ampicillin (100 µg ml–1), kanamycin (100 µg ml–1) or rifampicin (200 µg ml–1) added as required. At. caldus strains were grown at 37 °C in tetrathionate medium (3 mM), sterilized and adjusted to pH 2·5, as reported previously (Rawlings et al., 1999).


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Table 1. Bacterial strains and plasmids

 
DNA isolation and manipulations.
Plasmid preparation, restriction endonuclease digestion, gel electrophoresis, ligation and Southern blot hybridization were performed using standard methods (Sambrook et al., 1989). Total DNA was extracted from At. caldus strain #6, which had been isolated from the arsenopyrite bio-oxidation plant at the Fairview mine, Barberton, South Africa. For the construction of the At. caldus strain #6 gene bank containing large inserts, chromosomal DNA was isolated as follows. At. caldus cells were harvested by centrifugation, washed three times in acidified water (pH 1·8), and resuspended in TE buffer, pH 7·6. Lysis was with 1 % SDS in the presence of proteinase K (1 mg ml–1) at 37 °C. DNA was precipitated with ethanol, washed twice in 70 % ethanol and resuspended in TE buffer (pH 7·6). This DNA was partially digested with Sau3A, the fragments separated using a sucrose gradient and fragments in the 10–25 kb size range were ligated into the BglII site of the positive selection cloning vector, pEcoR252. Approximately 9600 colonies were obtained by transforming the ligation mix into E. coli DH5{alpha} and selecting for growth on Luria agar (LA) plus ampicillin (100 µg ml–1). These colonies were scraped from an LA plate and used to prepare the At. caldus gene bank. For the construction of plasmid containing the entire ars operon on one continuous fragment, the following cloning strategy was employed. A 7 kb BamHI–ClaI fragment from pArs1 was ligated to a 6·4 kb ClaI–PstI fragment from pAtcars3.2, and the resulting 13·4 kb fragment was in turn cloned in the BamHI–PstI sites of the cloning vector pEcoR252, resulting in pTnArs. Sequencing was by the dideoxy chain-termination method, using an ABI PRISM 377 automated DNA sequencer; the sequence was analysed using a variety of software programs but mainly the PC based DNAMAN (version 4.1) package from Lynnon BioSoft. Comparison searches were performed using the gapped-BLAST program at the National Centre for Biotechnology Information.

PCR.
PCR was performed with primers described in Table 2. The reactions were carried out in a Biometra thermocycler with an initial denaturation at 94 °C for 60 s, followed by 25 cycles of denaturation (30 s at 94 °C), an annealing step of 30 s, and a variable elongation step at 72 °C. Annealing temperatures and elongation times were altered as required.


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Table 2. Primers

 
Assay for transposition.
The insert from plasmid pTnArs was cloned into the non-mobilizable cloning vector, pUC19, resulting in pArsTnpUC1 (Apr). pArsTnpUC1 was transformed into E. coli XL-1 Blue containing the conjugative plasmid pSa (Kmr). A minimum of three transformants were picked, inoculated into 5 ml LB, and incubated overnight to allow transposition to occur. The transformants were mated with rifampicin-resistant E. coli ACSH50Iq. Matings were carried out by mixing equal volumes of donor and recipient strains on the surface of an LA plate, and incubating overnight. The growth was scraped off the plate, resuspended in LB, and suitable dilutions were spread onto selective media: LA plus rifampicin (selection against the donor), LA plus rifampicin and kanamycin (selection for transformants) and LA plus rifampicin, kanamycin and 1 mM arsenate (selection for the transposon). Plasmid DNA was prepared from individual colonies, digested and analysed for the presence of the TnAtcArs insertion by pulsed-field gel electrophoresis and Southern blot hybridization. A 900 bp KspI–HindIII arsB DNA fragment from plasmid pArs38 was used as a probe.

Pulsed-field gel electrophoresis.
Digested DNA was set in an equal volume of 2 % LMP agarose (Seaplaque, FMC Bioproducts). Trans-alternating field electrophoresis (TAFE) was performed using a Beckman GeneLine apparatus. DNA fragments were separated in a 1 % agarose (SeaKem LE, FMC Bioproducts) gel at 100 mA and 12 °C for 16 h with a pulse interval of 5 s.

Arsenic resistance assays.
To test for growth of At. caldus in the presence of arsenite, cells were cultured in the tetrathionate medium described above containing 0, 20 or 30 mM arsenite. Actively growing cultures were diluted 100-fold into fresh media and incubated for 20 days, and the OD600 was determined. Growth in the presence of arsenate was not tested, as the phosphate concentration in the growth medium would contribute to arsenate resistance (Silver et al., 1981). Assays performed in E. coli ACSH50Iq containing plasmids were carried out in LB medium containing appropriate antibiotics and various concentrations of sodium arsenite. Growth assays to determine the resistance to arsenate were performed in low-phosphate medium (Oden et al., 1994) supplemented with 2 mM K2HPO4. Overnight cultures were diluted 100-fold into fresh medium and incubated at 37 °C for 5 h, and the OD600 was determined. The incubation time used corresponds to the mid-exponential growth phase of controls under the same conditions. In all cases the resistance was expressed as the percentage OD600 compared with the control culture with no added arsenic.

Analysis of the arsenate reductase mechanism.
The plasmid pArs1GL, which has the 13·4 kb insert from pArs1 cloned in pGL10, was constructed and was used to test for arsenate resistance in the E. coli BH5262 double mutant. Cells were also transformed with pTrx6 (Table 1). To determine resistance to arsenate, cells were plated on media containing 0, 2 and 5 mM sodium arsenate and were incubated overnight at 37 °C.

RNA analysis and RT-PCR.
Total RNA was isolated as described previously (Trindade et al., 2003) from 50 ml of mid-exponential-phase cultures of E. coli ACSH50Iq carrying various plasmids, grown in LB containing 0·1 mM arsenate, 0·1 mM arsenite or no arsenic, and with antibiotic selection. RNA was also isolated from At. caldus #6. Cells were first grown in tetrathionate medium without arsenite, and were then diluted 100-fold into fresh medium containing 0·1 mM arsenite or no arsenite. RNA was isolated using the same method as for E. coli. The RNA was separated on a 1 % formaldehyde denaturing gel by standard procedures (Sambrook et al., 1989), and transferred to Hybond-N+ nylon membrane (Amersham) and hybridized according to the manufacturer's instructions, using digoxigenin-labelled DNA probes specific for the arsA and tnpA transcripts. The probes were synthesized using either the PCR DIG Probe Synthesis Kit or the Random Primed DNA Labelling Kit (Roche), using the primer combination RNAarsAF/RNAarsAR for arsA and TnpAF/pEcoF for tnpA (Table 2).

For RT-PCR, the 1st Strand cDNA synthesis kit (AMV; Roche) was used for cDNA synthesis and cDNA product detection. The protocol of the manufacturer was used for the reverse-transcriptase reaction. The PCR was performed as described above, using 2 µl of the 20 µl (total volume) reverse-transcriptase reaction, and the extension times were altered as required for the different primer pairs. The TnpART and ArsBRTR primers were used for cDNA synthesis from tnpA and arsB mRNA, respectively (Table 2, Fig. 1), and were used in combination with the TnpALacZF and RNAorf8F primers for the PCR, respectively. As a positive control for tnpA and arsB cDNA synthesis, the TnpAF and RNAarsBF primers, respectively, were used for the PCR. To detect DNA contamination in the mRNA extracts, reactions were performed with each primer pair without any AMV reverse transcriptase.



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Fig. 1. Physical and genetic map of TnAtcArs from At. caldus #6 showing the location of the genes identified and the subclones used in this study. The X in construct pArs1{Delta}DA7 indicates a frame-shift mutation that was introduced in ORF7 by blunting the ClaI site and religating. IR, inverted repeats. The locations of primers TnpALacZF, TnpAF, TnpART, ArsBRTR, and RNAorf8F (Table 2) used for the RT-PCR analysis are shown. The * indicates a methylated restriction enzyme site.

 
Construction of the promoter–lacZ fusions.
The putative promoter regions for arsR, tnpR and tnpA were amplified by PCR using the following primer pairs: ArsRLacZR2/TnpRLacZR (600–931 bp) for arsR and tnpR, and TnpALacZF/TnpALacZR2 (9193–9579 bp) for tnpA (Table 2). The PCR products were digested with either BamHI or BamHI/EcoRI and ligated to the promoterless lacZ reporter gene of pMC1403. For the arsR–tnpR promoter region the PCR product could be cloned directionally, resulting in an arsR–lacZ fusion in the one direction and a tnpR–lacZ fusion in the other direction. Fusions were confirmed by DNA sequencing.

{beta}-Galactosidase assays.
Overnight cultures were diluted 1 : 200 into fresh medium containing the appropriate antibiotics, 0·4 mM IPTG, sodium arsenate or sodium arsenite (each 25 µM) when indicated, and incubated at 30 °C to an OD600 of 0·5. The {beta}-galactosidase activities were measured using the method of Miller (1972).


   RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
At. caldus growth experiments in arsenite
The growth rates of six At. caldus strains were tested in the presence of arsenite. Three of these strains, #6, f and MNG, were isolated from arsenic-containing environments and three from environments not known to contain arsenic. After 6 days growth in the presence of 20 mM arsenite At. caldus strains #6, f and MNG had reached stationary phase whereas strains BC13, C-SH12 and KU were still in lag phase. Furthermore, growth of strains, #6, f and MNG was always obtained in the presence of 30 mM arsenite within a 20 day period, whereas growth for strains BC13, C-SH12 and KU was inhibited at this concentration (not shown).

Cloning of the entire transposon, TnAtcArs
We had previously isolated three small fragments of the At. caldus ars-containing operon (de Groot et al., 2003); however, we were unable to link the small fragments into one continuous piece. A gene bank of At. caldus strain #6 was therefore prepared that contained inserts greater than 10 kb in size. The large-insert-containing At. caldus gene bank was screened for arsenite and arsenate resistance in the E. coli ars deletion strain ACSH50Iq. Plasmids were isolated from colonies that grew on 0·5 mM arsenite and 1·0 mM arsenate. These plasmids were retransformed into E. coli ACSH50Iq to confirm their ability to confer arsenic resistance and the larger of these plasmids were mapped. All these plasmids were truncated in the tnpA gene (see below). Using the cloning strategy described in Methods, the entire ars operon was cloned as one continuous fragment, pTnArs.

Sequence analysis of TnAtcArs
The insert of plasmid pTnArs was sequenced in both directions and eleven ORFs were identified. Nine of these were transcribed in the same direction, and were located between the divergently transcribed tnpR and tnpA genes (Fig. 1). Several features typically associated with transposons were identified (discussed below) and therefore the name TnAtcArs was given. Analysis of the tnpR and tnpA genes revealed that the TnAtcArs clearly belongs to the large Tn3 family of transposons, with some features that are more similar to the Tn3 subfamily and others that are closer to the Tn21 subfamily (Sherrat, 1989). As in Tn3, the tnpA (transposase) and tnpR (resolvase) genes are divergently transcribed, with a res site (recombination site) located between them immediately upstream of tnpR, whereas in the Tn21 subfamily of transposons the tnpR and tnpA genes are transcribed as a unit, with the res site upstream of both genes (Grinsted et al., 1990). However, the nucleotide sequences of the res-like sites (Fig. 2a) are more closely related to Tn21 than to Tn3. The left and right IR sequences of TnAtcArs are 40 bp long and identical to each other (Fig. 2b). They are also 37/38 bp identical in sequence to the Tn21 left IR and 34/38 bp identical to the Tn21 right IR, but only 21/38 bp identical to the Tn3 IR sequences (left or right). The amino acid sequence of the resolvase is most closely related to Tn5037 (Kaliaeva et al., 2001), a transposon that is most closely related to the Tn21 subgroup, while the transposase is most closely related to that found in Alcaligenes faecalis (Table 3) and is also a member of the Tn21 subgroup.



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Fig. 2. (a) Sequence of the arsR–tnpR intergenic region, showing the primers used for the lacZ fusions (underlined) and the translational starts (arrows). The tnpR res sites are shown in bold. (b) Alignment of the inverted repeat sequences of TnAtcArs, Tn21 and Tn3. The underlined sequences represent the bases that are different compared to the Tn21 left repeat sequence, showing that the TnAtcArs sequence is more similar to Tn21 than to the Tn3 subgroup.

 

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Table 3. Locations and sizes of ORFs on TnAtcArs

 
The seven ORFs located between the tnpR and tnpA genes had high amino acid sequence identity to gene products associated with arsenic resistance (Table 3). Unexpectedly, the arsD and arsA genes were present as tandem duplicate copies that were almost identical, except that the product of the second copy of arsA was 12 aa shorter than that of the first copy. A 2122 bp duplication in DNA sequence had occurred, but it was not possible to determine exactly where within the two repeated regions the duplication had occurred. Two ORFs that have not been previously associated with arsenic resistance were situated between the second copy of arsA and the arsB gene (Fig. 1). The first of these ORFs encoded a product 470 aa in size with highest sequence relatedness to a 466 aa NADH oxidase from Lactobacillus plantarum (Table 3) and to NADH oxidases from other organisms. The second ORF not usually associated with ars genes encoded a 158 aa protein with identity to CBS-binding domains of several types of proteins. CBS (cystathione-{beta}-synthase) domains are small domains of unknown function that usually occur in two to four copies per protein and dimerize to form a stable globular structure. Some proteins that contain CBS domains may play a regulatory role (Bateman, 1997).

The TnAtcArs ArsR does not have the typical ELCVCDL metal-binding domain that has been experimentally determined (Shi et al., 1994), but is more closely related to a subgroup of ArsR proteins that was identified in a previous study (Butcher & Rawlings, 2002). Bacterial ArsC proteins are divided into two families. The first type are those such as the Staphylococcus aureus pI258 and Bacillus subtilis ArsC proteins that require thioredoxin to function and have been shown to require three essential cysteine residues, Cys 10, 82, 89 (Bennett et al., 2001; Messens et al., 2002). The second type of reductases require glutathione and glutaredoxin; within this group the E. coli R773 ArsC has been shown to have a catalytic cysteine, Cys-12, in the active site surrounded by an arginine triad composed of Arg-60, 94 and 107 (Martin et al., 2001). The TnAtcArs ArsC groups with the thioredoxin-requiring reductases and has the three essential cysteine residues with spacing typical of this family.

The arsDA duplication is absent from some At. caldus strains
To establish whether the arsDA duplication was present in all of the arsenic-exposed strains, #6, f and MNG, a Southern hybridization was performed, using a 1·4 kb NcoI–KspI fragment as a probe, containing the arsDA genes from At. caldus #6. Genomic DNA was digested with NcoI, which cuts once within the arsA gene; where a tandem arsDA duplication has occurred this will result in a fragment of approximately 2·2 kb. A signal of approximately 2·2 kb was obtained for At. caldus strains #6 and MNG (Fig. 3), but not for strain f, whereas the sizes of the 1·2 kb (arsA-ORF7) signal and the larger 6·5 kb signal were similar for all strains. This result is what would be predicted if strain f did not have an arsDA duplication. Strains #6 and MNG give an additional signal which belongs to a truncated non-functional arsDA-containing operon, which has been isolated from At. caldus #6 (Tuffin et al., 2004).



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Fig. 3. Southern hybridization analysis of chromosomal DNA isolated from the six strains of At. caldus shown, digested with NcoI endonuclease and probed with a 1·4 kb NcoI–KspI DNA fragment from pArs1. The 2·2 kb fragment shown indicates a duplication of the arsDA genes.

 
TnAtcArs is functional in E. coli
To test whether the transposon was active in E. coli, cells containing the non-mobilizable plasmid pArsTnpUC1 and the conjugative plasmid pSa were mated with E. coli ACSH50Iq-Rif recipient, and plated on selective media. If the arsenic-resistant TnAtcArs was transposed into pSa it would be able to be mated into the E. coli recipient strain whereas pArsTnpUC1 would not be able to be conjugated into the recipient. Asr Kmr and Ass Kmr colonies were isolated and plasmid DNA from these transconjugants was analysed by Southern hybridization using arsB from pArs1 as probe (Fig. 4a). Plasmids isolated from Asr Kmr transconjugants hybridized to the arsB probe, while plasmids from the Ass Kmr transconjugants did not. This suggested that in Asr Kmr cells the transposon had jumped to pSa and was conjugated into the recipient cells, while in Ass Kmr cells, only pSa was transferred to the recipient. To further confirm transposition, restriction enzyme analysis of one of the transposon-containing plasmids as well as the original pSa plasmid, was carried out using a TAFE (trans-alternating field electrophoresis) gel (Fig. 4b). On digestion with BamHI and BglII, neither of which have cutting sites in the transposon, pSa produced fragments of 9 and 26 kb (lane 3) and 6, 8 and 22 kb (lane 5) respectively, while the size of the largest of these fragments was increased following the insertion of the transposon (lanes 2 and 4). Further digests with restriction enzymes that do cut within the transposon (SalI, NcoI and KspI), were performed to confirm the presence of the arsenic-resistance operon. These digests further indicated that a 2·2 kb fragment, containing a SalI and an NcoI site, had been spontaneously deleted on transposition. PCR analyses indicated that only one copy of arsA and arsD was present. Twelve other transconjugants were analysed and all were found to have the second copy of arsDA deleted. The duplication of arsDA, therefore, appeared to be unstable following transposition and conjugation. These transconjugants were sensitive to ampicillin, demonstrating that they were not co-integrates of the pSa and pArsTnpUC1 plasmids. However, other Asr Kmr transconjugants that were resistant to ampicillin were also isolated. Analysis of their plasmids indicated the presence of both pSa and ArsTnpUC1, but they were not analysed further.



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Fig. 4. Transposition of TnAtcArs in E. coli. (a) Southern hybridization of plasmids isolated from transconjugants after the mating procedure. Plasmid DNA was digested with BglII and hybridized with arsB from pArs1. Lanes 1–4, plasmids from transconjugants containing the transposon (pTn1–4); lanes 5–6, plasmids from transconjugants containing pSa; lane 7, pSa; lane 8, pArsTnpUC1. (b) Restriction digests of plasmids pTn1 and pSa with BamHI (lanes 2 and 3) and BglII (lanes 4 and 5) showing increase in size of pSa fragments after TnAtcArs transposition. Lanes 1 and 6, {lambda} DNA digested with HindIII and BglII respectively as molecular mass markers.

 
Arsenic resistance of TnAtcArs and ars deletion constructs in E. coli
The ability of the construct pArs1 to confer resistance to arsenite and arsenate was tested in E. coli ACSH50Iq. Resistance was compared to the E. coli R773 arsenic genes (pUM3). E. coli ACSH50Iq cells containing pArs1 were considerably more resistant to both arsenite and arsenate than cells containing vector only, but slightly less resistant than cells containing the cloned E. coli arsRBC genes present on pUM3 (Fig. 5). In an attempt to discover the effect of the arsDA duplication, plasmid pArs1{Delta}DA was made from construct pArs1 (where one of the duplicate arsDA copies was removed by making a 2·2 kb KspI deletion), and cells containing it were assayed for arsenic resistance. Cells containing pArs1{Delta}DA were not significantly more or less resistant to either arsenite or arsenate than pArs1. A further construct, pArs1{Delta}DA78, which contained a 4·1 kb KspI deletion that extended from the beginning of the second arsD to the end of ORF8, was constructed, and its effect on the growth of E. coli ACSH50Iq was also examined. There was no marked difference in resistance compared to cells with pArs1 in the presence of arsenic (Fig. 5). A similar result was obtained with plasmid pArs1{Delta}DA7, which had one copy of arsDA deleted and had a frame-shift mutation introduced at the ClaI site in ORF7 (Fig. 1 and data not shown). A problem experienced in these experiments was that spontaneous arsDA deletions occurred in a varying proportion of cells, and so the deletion constructs were designed to only have a single copy of arsDA. The occurrence of these spontaneous deletions indicated that these arsenic resistance results should not be overinterpreted. Nevertheless, the deliberate deletion of either arsDA or ORFs 7 and 8 did not appear to have a marked effect on arsenic resistance in E. coli. To confirm these results, the growth rates conferred by these constructs in E. coli in media containing 1·5 mM arsenate were compared. This is a more accurate determination of resistance than an end-point analysis. As before, there were no significant differences in growth rates (data not shown), confirming that, at least in E. coli, these additional genes did not seem to play an important role in arsenic resistance.



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Fig. 5. Arsenic growth assays performed in E. coli ACSH50Iq in the presence of various concentrations of either arsenate or arsenite. Cell densities were determined (OD600) and percentage resistance was calculated (100 % was equivalent to an OD600 of approximately 0·5). x, pEcoBlunt; {circ}, pUM3; {square}, pArs1; {blacksquare}, pArs1{Delta}DA; {blacktriangleup}, pArs1{Delta}DA78. Each data point represents the results of at least two independent experiments. The error bars indicate standard deviations.

 
Arsenate reduction by products of the TnAtcArs arsenic-resistance operon
Although deletion of ORFs 7 and 8 had no effect on arsenic resistance in E. coli, we were interested in investigating whether they could play a role in the reduction of arsenate. An E. coli strain with mutations in the trxA (thioredoxin) and gshA (responsible for the synthesis of glutathione) genes, transformed with pArs1GL, was tested for arsenate resistance. The plasmid was, however, unable to confer arsenate resistance; therefore, the products of ORFs 7 and 8 did not appear to encode an NADH-dependent arsenate reduction mechanism that could alleviate the requirement for thioredoxin or glutathione in E. coli. When the E. coli mutant was transformed with pArs1GL, together with a plasmid containing the thioredoxin gene from Acidithiobacillus ferrooxidans (pTrx6), some resistance to 2 mM sodium arsenate was restored. Cells containing only pTrx6 remained sensitive to arsenate. As discussed above, TnAtcArs ArsC is most similar to the thioredoxin-utilizing reductases, and these results give experimental evidence to suggest that TnAtcArs ArsC does function by using thioredoxin.

The tnpA gene is transcribed separately from the ars operon
Northern blot hybridization was performed to determine whether the arsenic-resistance genes were transcribed as an operon, and whether the tnpA gene was transcribed separately. RNA was isolated from E. coli ACSH50Iq transformed with pArs1, pArsTnpUC1 and p{Delta}Ars grown in medium in the presence or absence of arsenite. The RNA was hybridized to probes specific for arsA and tnpA. A transcript starting at approximately 10 kb was obtained from cells containing pArs1 and pArsTnpUC1 induced with arsenite, while no signal could be detected when cells were uninduced (Fig. 6). A very weak signal was obtained from cells grown in the presence of arsenate (not shown). This suggests that the arsenic-resistance genes, presumably from the arsR to the end of arsB, are co-transcribed as an operon, and that expression from the arsR promoter is positively regulated by the presence of arsenate, and even more so by arsenite. RNA extracted from At. caldus #6 was also analysed by Northern hybridization but a large transcript could not be detected, presumably because the mRNA was degraded as a result of its slow growth rate. When hybridized with the tnpA probe, a transcript starting at approximately 1·4 kb was obtained from cells containing pArs1 and p{Delta}Ars (both of which do not have a full-length tnpA gene), and the signal intensity was the same for RNA extracted from cultures grown in the presence and absence of arsenic; however, no tnpA transcript was detected from samples containing ArsTnpUC1 (full-length). Taken together, these results suggest that tnpA was not transcribed as an operon with the arsenic-resistance genes, and probably has its own promoter, whose expression is not regulated by arsenic, but by the presence of a full-length tnpA. In order to confirm these results, RT-PCR was conducted, using RNA extracted from E. coli ACSH50Iq harbouring plasmids pArs1 and pArsTnpUC1, grown in the presence of arsenite, and also RNA extracted from At. caldus #6 grown in the presence of arsenite. The position of the primers used for this analysis is shown in Fig. 1. When using the ArsBRTR/RNAorf8F primer combination, an 855 bp product was obtained from the At. caldus sample and from the E. coli plasmid-containing samples, indicating that arsB was co-transcribed with orf8 (data not shown). When using the TnpART/TnpALacZF primer combination, a 552 bp product would be obtained only if tnpA was co-transcribed with arsB. While RT-PCR products specific to tnpA (120 bp) were detected using primers TnpART/TnpAF, a product of the arsB–tnpA intergenic region was not obtained. From this it was concluded that co-transcription of arsB and tnpA did not occur. Detection of the tnpA RT-PCR from the full-length tnpA (pArsTnpUC1) construct in spite of no product being detected by Northern hybridization suggested a low level of tnpA expression.



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Fig. 6. Northern blot of mRNA from E. coli ACSH50Iq carrying the plasmids pArs1 (truncated tnpA), pArsTnpUC1 (full-length tnpA) and p{Delta}Ars (truncated tnpA with 1340 bp upstream region) probed with DNA probes specific to the arsA and tnpA genes. RNA was isolated from cells grown in the absence (–) or presence (+) of 100 µM sodium arsenite. The positions and approximate sizes of the transcripts are shown.

 
Reporter-gene studies to investigate regulation of putative promoter regions
To study expression from the three putative promoters, translational lacZ fusions were made, where 10, 38 and 7 aa of the ArsR, TnpR and TnpA proteins, respectively, were included (Fig. 2a). For the TnpA fusion, 363 bp upstream of the tnpA ATG start were included. With each of the promoter fusions, constructs expressing ArsR, ArsD or TnpR from a tac promoter were added in trans (Table 1, Fig. 7). When the arsR–lacZ constructs were expressed in E. coli ACSH50Iq in the absence of arsenic, {beta}-galactosidase activities of approximately 1600 units were obtained (Fig. 7a). Activity dropped over fourfold to approximately 400 units when ArsR expressed from the tac promoter was added in trans, and this repression was completely relieved when either sodium arsenate or sodium arsenite was added. This indicated that the arsR gene product negatively regulated expression of the arsenic-resistance operon and that arsenite and arsenate were inducers. The presence of ArsD in trans had no effect on {beta}-galactosidase activity. Since the arsR promoter reads divergently from the tnpR promoter, the promoters could overlap and the TnpR repressor might affect expression in both directions. We therefore wished to test whether TnpR would have any regulatory effects on the expression of the arsenic genes. Expression of TnpR from the tac promoter in trans did not have any effect on the {beta}-galactosidase activity, suggesting that TnpR did not affect expression from the arsR promoter.



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Fig. 7. {beta}-Galactosidase activity measured in E. coli ACSH50Iq carrying different promoter constructs in the presence of 25 µM sodium arsenate and arsenite. (a) ArsRLacZ fusion construct with the following plasmids in trans: ptacGL control plasmid (white bars); ptacArsR (black bars); ptacArsD (stippled bars); and ptacTnpR (hatched bars). (b) TnpRLacZ fusion construct with the following plasmids in trans: ptacACYC control plasmid (white bars); ptacTnpR (black bars); and ptacArsR (hatched bars).

 
Similar expression studies with the tnpR–lacZ fusion indicated that the presence of TnpR in trans reduced {beta}-galactosidase activity 2·2-fold, from approximately 1300 to 600 units (Fig. 7b). Neither the presence of arsenic, nor the presence of ArsR expressed from the tac promoter in trans, had an effect on expression from the tnpR promoter. Therefore, TnpR autoregulates its own expression, and this is not affected by the presence of arsenic or ArsR. Although the tnpA–lacZ fusion gave faint blue colonies on agar containing X-Gal, no activity could be measured within a 2 h {beta}-galactosidase assay. Previous reports have shown that some tnpA promoters are very weak (Hõrak & Kivisaar, 1999); however, as tnpA transcript was easily detected, promoter activity should also have been detected by reporter gene expression. The most likely explanation is that a region more than 363 bp upstream of the tnpA ATG is required for promoter activity. Additional studies showed that when the 363 bp upstream region and a 1340 bp upstream region were cloned in a promoterless CAT (chloramphenicol acetyltransferase) expression vector, resistance to Cm was obtained with the larger upstream region only (data not shown).


   DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
TnAtcArs is a transposon that appears to have evolved specifically for the purpose of distributing the arsenic-resistance genes that it carries. Not only the presence of the ars genes but also the structure of the transposon suggests this. In most transposons of the Tn21 subfamily, the tnpR (resolvase) and tnpA (transposase) genes are physically adjacent to each other (Grindsted et al., 1990; Liebert et al., 1999). In this way, whatever other genes the transposon may accumulate, these two transposon-associated genes remain together so that they can operate as a unit. In the case of TnAtcArs, the tnpR and tnpA genes have become separated by a region of approximately 10 kb that contains the ars genes. It is as though the tnpR and tnpA genes have enclosed the ars genes in such a way that the transposon has become dedicated to the movement of these genes.

Sequence data indicated that the gene for the transposase was located downstream of the arsB gene and would be transcribed in the same direction as the ars genes. This raised the question of whether transcriptional read-through from the ars genes may increase the level of expression of the tnpA gene and hence the frequency of transposition in response to arsenic. Increased expression of transposase has been shown to result in an increase in transposition of insertion sequence IS2 (Lewis et al., 2004). An increased frequency of transposition in response to mercury has also been reported in the mercury-resistance transposon Tn501 (Schmitt et al., 1981). Although transposition of TnAtcArs was observed in E. coli, we were unable to detect an increase in the frequency of transposition in response to arsenic (data not shown). This was due mainly to a large scatter in the transposition frequency. The Northern hybridization data showed that tnpA was not affected by the presence of arsenite, and therefore increased transposition in response to arsenite would not have been expected.

The ars genes present on TnAtcArs have several atypical features that have not been reported in other ars systems. One of these is the duplication of the arsD and arsA genes. Studies on the effect of the arsDA duplication on arsenic resistance were complicated by the observation that the duplication appeared to be unstable in E. coli. Spontaneous deletion of one of the arsDA copies took place in a variable proportion of cells during the course of a growth experiment. Furthermore, all TnAtcArs transposons that were captured by transposition onto pSa had only a single copy of arsDA. Whether this deletion took place during the transposition step or during conjugal transfer to the recipient cells is not certain. The arsDA duplication is possibly more stable in At. caldus, as two of the three industrial strains studied appeared to contain the 2·2 kb duplication. From an analysis of TnAtcArs containing the arsDA deletion it was established that the second arsA gene encoding the smaller ArsA protein of 600 aa was the copy that was retained, but the site of duplication could lie anywhere within the 2·2 kb repeat region. The mechanism of arsDA duplication is unknown. Furthermore, although we used TnAtcArs arsDA deletion transposons in many experiments we never observed duplication of the arsDA genes in E. coli. The role of the arsDA duplication and the possible reasons for the presence of additional copies of an ArsA ATPase or the ArsD repressor of ars gene overexpression are not obvious. It has been observed that the arsD and arsA genes nearly always occur together and that the two proteins functionally interact, suggesting that they operate together in arsenic detoxification (B. P. Rosen, personal communication). However, it is not clear how additional copies of arsDA would produce an increase in arsenic resistance without additional copies of the ArsB exporter.

A second atypical feature of the ars genes is that two ORFs (ORFs 7 and 8) were present between arsA and arsB, and such ORFs have not previously been associated with arsenic resistance. These ORFs had clear sequence identity to an NADH oxidase and a protein containing a CBS domain (Table 3). The effect of ORFs 7 and 8 on arsenic resistance was not clear. To reduce possible variability associated with the spontaneous deletion of one of the copies of arsDA, we deleted both ORFs 7 and 8 from a construct in which a second copy of arsDA had already been deleted (Fig. 1). Growth analyses in various concentrations of both arsenate and arsenite indicated that cells with the deletion constructs were equally resistant to both forms of arsenic compared to cells without the deletion of ORFs 7 and 8 (Fig. 5). To exclude the possibility that the NADH oxidase-like protein (ORF7) was associated with the reduction of arsenate, the ORF7- and ORF8-containing constructs and their deletions were tested for their ability to complement an E. coli trxA (thioredoxin) gshA (responsible for the synthesis of glutathione) double mutant. Complementation was not achieved, and therefore the gene product of ORF7 (or ORF8) did not seem to be involved in an alternative arsenate reduction system. These deletion analyses were performed in a heterologous host and it is possible that the effects of these genes might be masked in E. coli.

As the divergent promoters of tnpR and arsR overlap (Fig. 2a), it was of interest to examine whether the interaction of a regulator in the promoter region of one of the genes affected transcription from the other. Northern hybridization analysis suggested that the ars genes were transcribed as a single 10 kb mRNA fragment (Fig. 6) that corresponded in size to a transcript extending from the start of arsR to the end of arsB. Furthermore, this transcript could only be detected if arsenite was present in the medium. An arsR–lacZ reporter fusion confirmed ArsR-regulated, arsenite- and arsenate-induced expression from the arsR gene (Fig. 7a). It was interesting that no regulation of the arsR–lacZ fusion by ArsD was detected when ArsD was expressed from a plasmid placed in trans. Also no interference of ArsR on expression of tnpR was detected, as arsenic had no effect on tnpR–lacZ reporter gene activity in the presence or absence of ArsR. The tnpR–lacZ reporter indicated that expression of the tnpR–lacZ fusion protein was repressed by TnpR when expressed from a tac promoter and located on a plasmid placed in trans (Fig. 7b). The Northern hybridization (Fig. 6), RT-PCR (not shown) and tnpA–lacZ fusion experiments suggested that expression of tnpA was controlled by a promoter independent of the ars genes, and that its expression was unaffected by arsenic. An apparent anomaly is that transcription of tnpA was easily detected by Northern hybridization (Fig. 6) when a truncated tnpA gene was used, whereas when the complete tnpA gene was present no tnpA transcript could be detected. Transposon transposition is potentially lethal, and cells maintain precise control over the amount and activity of transposase, and the regulatory mechanisms act at various stages of the transposition process (Nagy & Chandler, 2004). The results obtained in this study suggest that either the full-length tnpA transcript or the TnpA product itself is involved in the regulation of tnpA at the transcriptional level, and that the tnpA deletion has lost this regulation activity. As mentioned above, the control of Tn3 and Tn21 transposition is by repression by TnpR, which binds to the res sites in the promoter region (Grinsted et al., 1990), and the TnAtcArs TnpR could similarly regulate tnpA expression. However, no res sites to which TnpR is expected to bind were identified upstream of tnpA. Detailed analysis of tnpA expression beyond whether it was linked to expression of the ars operon was not the aim of this study and it was decided not to pursue this further.

A characteristic of commercial arsenopyrite bio-oxidation systems is that they are non-sterile, continuous-flow processes. Crushed mineral concentrate, water and small quantities of low-grade fertilizer are continually added, all of which might contain new organisms (Dew et al., 1997). It is of interest to know from where the highly arsenic-resistant At. caldus isolates might have acquired TnAtcArs. Recently the sequence of a set of arsR, arsC, arsD, arsA, arsB and tnpA genes from Alcaligenes faecalis has been deposited in the GenBank database (accession no. AY297781). These genes are by far the most closely related to the equivalent genes from TnAtcArs (Table 3) based on nucleotide and amino acid sequence as well as gene order. Differences are that the equivalent of ORF7 (NADH oxidase-like protein) is missing and ORF8 (CBS domain protein) is much smaller. There is also a highly homologous tnpA gene downstream of arsB, indicating that the Al. faecalis ars genes are also associated with a Tn21-like transposon. However, tnpR and one IR sequence are missing and therefore the transposon is probably defective. Nevertheless, because the two sets of ars genes are so similar, they must have evolved from a common ancestral transposon. How these have been horizontally transferred such that one set is present in a heterotrophic neutrophilic bacterium such as Al. faecalis and another in a highly acidophilic sulphur-oxidizing acidophile such as At. caldus is unclear. Although we may never know from where the TnAtcArs set sail before arriving in At. caldus, this study has provided supporting evidence for the view that led to Tn21 being called the ‘flagship of the floating genome’ (Liebert et al., 1999).


   ACKNOWLEDGEMENTS
 
We thank Kevin Hallberg for At. caldus strains BC13, KU and C-SH12, and Bronwyn Butcher for E. coli ACSH50Iq. This work was funded by grants from the National Research Foundation (Pretoria), The Human Resources for Industry Program (THRIP, Pretoria), the University of Stellenbosch and the BHP-Billiton Johannesburg Technology Centre.


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Received 18 April 2005; accepted 8 June 2005.



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