Department of Microbiology, Stellenbosch University, Private Bag X1, Matieland, 7602, South Africa
Correspondence
Douglas E. Rawlings
der{at}sun.ac.za
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ABSTRACT |
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INTRODUCTION |
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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 l1 and arsenite concentrations of 36 g l1 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.
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METHODS |
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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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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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-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 -galactosidase activities were measured using the method of Miller (1972)
.
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RESULTS |
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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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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 NcoIKspI 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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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 pArs 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
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 arsBtnpA 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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DISCUSSION |
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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 arsRlacZ reporter fusion confirmed ArsR-regulated, arsenite- and arsenate-induced expression from the arsR gene (Fig. 7a
). It was interesting that no regulation of the arsRlacZ 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 tnpRlacZ reporter gene activity in the presence or absence of ArsR. The tnpRlacZ reporter indicated that expression of the tnpRlacZ 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 tnpAlacZ 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
).
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ACKNOWLEDGEMENTS |
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Received 18 April 2005;
accepted 8 June 2005.
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