Department of Microbiology, University of Stellenbosch, Private Bag X1, Matieland 7602, South Africa1
Author for correspondence:Douglas E. Rawlings. Tel: +27 21 808 5848. Fax: +27 21 808 5846. e-mail: der{at}sun.ac.za
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ABSTRACT |
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Keywords: arsenic resistance, ArsR-like proteins, gene regulation
a Present address: Department of Biology, Williams College, Williamstown, Massachusetts 01267, USA.
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INTRODUCTION |
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The arsenic-resistance (ars) operon from At. ferrooxidans was found to consist of two divergent elements, arsRC and arsBH. The genes within these elements were shown to confer arsenic resistance upon an Escherichia coli ars mutant, AW3110 (Butcher et al., 2000 ). However, arsH was not required for arsenic resistance in E. coli and it is not yet known whether it is required in At. ferrooxidans. The unusual divergent arrangement of the ars operon of At. ferrooxidanshas raised questions about the regulation of its genes. The finding that the arsB and arsC genes are located on divergent elements may mean that they are independently regulated. The putative ArsR protein from At. ferrooxidans only had weak homology to ArsR proteins from well-studied arsenic-resistanceoperons and did not contain the conserved metal-binding box, ELCVCDL, to which the arsenite inducer binds (Shi et al., 1994
, 1996
). Therefore, it was unclear whether the putative ArsR protein coded for within the At. ferrooxidans operon was able to regulate these genes in response to arsenite.
Here we describe an investigation into the regulation of the chromosomal ars operon of At. ferrooxidans and into whether the ArsR-like protein of this organism is a regulator of the ars operon in response to arsenite, arsenate and antimonite.
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METHODS |
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Construction of the arsenic-sensitive lac-negative E. coli strain.
The generalized transduction method using phage P1vir (Miller, 1972 ) was used to construct this strain. Phage P1vir was isolated from E. coli AW3110 (Carlin et al., 1995
) and used to transduce E. coliCSH50Iq. The transduced cells were selected on chloramphenicol and X-Gal plates. They were then checked for sensitivity to arsenic on plates containing 0·5 mM sodium arsenite. Sensitivity indicated that the
ars::cam from E. coli AW3110 had replaced the ars genes from E. coli CSH50Iq, resulting in strain E. coli ACSH50Iq.
Construction of the promoterreporter gene (lacZ) constructs.
The putative promoter regions were amplified by PCR using primer pairs BBARSB/BLACZE (arsB promoter) and RLACZB/RLACZE (arsR promoter) (Table 1). The resulting PCR products were digested with BamHI and EcoRI (the restriction sites of these enzymes were included in the primers; Table 1
) and ligated to the promoterless lacZ gene of pMC1403, which had been digested with the same enzymes; this resulted in pB3lacZ and pRlacZ, respectively, which were translational fusions of arsB and arsR with the lacZ gene (Fig. 1
). Primers BBARSB/BBARSC were used to amplify a region containing the arsB promoter and the intact arsRgene (Fig. 2
). This PCR product was digested with BamHI and cloned into pMC1403, which had been digested with BamHI. The product could be cloned in either direction. Cloning the insert in one direction resulted in pB2lacZ, in which the arsB promoter was fused to lacZ of pMC1403 and the intact arsR gene was transcribed in the opposite direction (Fig. 1
). Cloning the insert in the other direction resulted in lacZ being fused in-frame at the start of arsC, resulting in pC2lacZ (Fig. 1
). In both constructs, the arsR promoter and the intact arsRgenewere also included. The above PCR product was also digested with BamHI and SmaI and cloned into pMC1403, which had been digested with the same enzymes. The resulting construct, pBlacZ, contained a translational fusion of arsB with lacZ and a partly truncated arsR gene in cis(Fig. 1
).
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ß-Galactosidase assays.
Overnight cultures were diluted 1:100 into fresh medium containing the appropriate antibiotics. The fresh cultures were grown at 30 °C until an OD600 value of between 0·3 and 0·4 was obtained. Twenty-five micromolar sodium arsenite, sodium arsenate or potassium antimonite was then added to the cultures as an inducer and the cultures were grown for a further 1 h, after which the ß-galactosidase activities of the cultures were measured using the method of Miller (1972) .
Isolation of total mRNA.
The yeast total RNA isolation protocol of Liang & Pretorius (1992) was used. RNA was isolated from E. coli ACSH50Iq strains carrying pTfarsCRBH-ACYC, which contains the complete ars operon from At. ferrooxidans. The cells were grown overnight in LB and then diluted 1:100 into fresh media containing 25 µM sodium arsenite or 50 µM sodium arsenate. The cells were grown at 30 °C for a further 4 h, prior to RNA being isolated from the cultures. RNA was also isolated from At. ferrooxidans ATCC 33020. At. ferrooxidans was first grown without arsenic in 800 ml tetrathionate medium. The cells from this culture were resuspended in 1·5 ml acid water and used to inoculate media containing 25 µM sodium arsenite, 25 µM sodium arsenate or no arsenic. The resulting cultures were incubated at 30 °C overnight and then used for RNA extractions.
Northern-blot analysis.
RNA (10 µg) from each sample was separated on a 1% denaturing agarose/formaldehyde gel. The gel was soaked in 20xSSC for 1 h and the RNA was then transferred to a Hybond-N nylon membrane under capillary-blotting in the presence of 20xSSC. The required probes (Fig. 1 and Table 1
) were labelled with [32P]dATP using the Random Primed DNA labelling kit (Roche Molecular Biochemicals) and hybridized to the RNA overnight at 60 °C in hybridization buffer (7% SDS, 1% BSA, 1 mM EDTA, 0·25 M Na2HPO4). After hydridization, the membrane was washed in 1xSSC/0·1% SDS and 0·1xSSC/0·1% SDS; it was then exposed to X-ray film to detect the bound probe.
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RESULTS |
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The sequences of the At. ferrooxidans ArsR protein and its nine closest matches clustered as a second subgroup of ArsR regulators upon homology analysis (Fig. 3b). Although all members of this subgroup shared conserved regions among themselves, and with SmtB and other known ArsR regulators, in the absence of a metal-binding domain, it was important to discover whether the atypical At. ferrooxidans ArsR protein was able to regulate the arsenic-resistance genes in response to arsenic.
arsBH and arsRC promoter studies
The arsenic-sensitive E. coli mutant strain AW3110 was wild-type for lac; hence, we constructed an E. coli strain (ACSH50Iq) that was both arsenic-sensitive and lac-negative. This ensured that there was no host cell background ß-galactosidase activity and that there were no E. coli chromosomal arsgenes present that may have interacted with the promoter-fusion constructs. E. coli ACSH50Iq was used for all of the work presented here; this strain also contained lacIqon the F' plasmid, ensuring that any genes that were added in trans and controlled by Ptac were repressed in the absence of IPTG.
To study the expression of the arsgenes we designed a number of translational gene fusions. The use of reporter genes to study gene expression should ideally be done in a homologous host, with a single gene copy integrated into the chromosome; however, this is not possible with At. ferrooxidans. To avoid many integration experiments into a heterologous E. coli host, all of the arsreporter gene fusions were transferred into the low-copy-number vector pGL10. Translational fusions were constructed where an intact arsR gene was included in cis (pB2lacZ-GL10 and pC2lacZ-GL10) and an arsBlacZ fusion was constructed in which 100 aa of ArsR were included (pBlacZ) (Fig. 1). An additional arsB translational fusion was constructed, in which the promoter region with only 71 aa of ArsR was included (pB3lacZ-GL10). Other fusions are shown in Fig. 1
.
Expression of arsBH
When the arsBlacZ constructs were expressed in E. coli ACSH50Iq in the absence of arsenic, ß-galactosidase activities of approximately 19 and 75 units, respectively, were obtained when the complete ArsR protein (pB2lacZ-GL10) and 71 aa of ArsR (pB3lacZ-GL10) were present (Fig. 4a). This indicated that in the absence of arsenic, the presence of an intact ArsR protein repressed the arsBH genes by approximately fourfold. With the addition of either 25 µM sodium arsenite or sodium arsenate this repression was relieved by three- to 3·5-fold provided that intact ArsR was present. When similar experiments were carried out with fusions in a higher-copy-number pMC1403-based vector, the presence of intact ArsR repressed arsBlacZ expression by about sevenfold (pB3lacZ vs pB2lacZ) in the absence of an inducer (Fig. 4b
). Following the addition of 25 µM sodium arsenite or sodium arsenate repression was lifted by about fivefold, while the addition of 25 µM potassium antimonyl tartrate reduced repression by about threefold (Fig. 4b
). An ArsR construct with 18 aa deleted from its C terminus (pBlacZ) gave similar levels of repression and derepression, respectively, in the absence and presence of arsenite, arsenate and antimony (Fig. 4b
). This indicated that the product of the arsR gene was able to regulate expression from ParsB and that while 100 aa of ArsR was sufficient for regulation of the promoter in response to arsenic and antimonite, the deletion of a further 28 aa inactivated ArsR. This additional deletion may have affected the ability of the At. ferrooxidans ArsR protein to dimerize, a feature that is essential for the functioning of the E. coli chromosomal ArsR protein and which was lost when more than 27 aa were deleted from the C terminus of this protein (Fig. 3
) (Xu & Rosen, 1997
).
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Interestingly, when the expression of pB3lacZ-GL10 was compared with the expression of pTfarsBH-ACYC and pTfarsB-ACYC in trans, there was a suggestion that arsH may play a regulatory role in arsB expression. However, attempts to investigate reporter gene expression with arsH expressed on its own from a Ptac indicated that ArsH did not affect tacarsR expression.
Expression of arsRC
When the arsRlacZ constructs were expressed in E. coli ACSH50Iq in the absence of arsenic in both a low-copy-number (pRlacZ-GL10) and a higher-copy-number vector (pRlacZ), the ß-galactosidase activities were 2 and 14 Miller units, respectively (Fig. 4a). These values were more than 15-fold less than the expression levels seen from the arsBlacZ fusion in equivalent vectors. The levels of expression detected from ParsR in the low-copy-number vector pRlacZ-GL10 were so low that further work was carried out only with reporter gene fusions in the higher-copy-number vector pRlacZ. As might be expected, the addition of arsenic to the arsRlacZ fusion did not result in an increase in reporter gene activity, presumably due to the absence of a functional ArsR protein (Fig. 4a
). Unexpectedly, the addition of combinations of the same ars genes, shown in Fig. 4(c)
, in trans did not affect the expression from pRlacZ in the presence or absence of arsenic (not shown). Therefore, it appears that if ArsR does autoregulate ParsR, either it is required in cis or there are elements downstream of the promoter that are required for induction by arsenic which were not included in pRlacZ.
An arsClacZ fusion was constructed that included arsR and its putative promoter region (pC2lacZ) and a second fusion was constructed that excluded the ParsR region (pClacZ). There was no detectable expression from pClacZ, which suggested that arsC does not have a promoter of its own but is transcribed together with arsR (results not shown). The arsClacZ fusion pC2lacZ, which contained an intact arsR gene, did respond to the addition of arsenic, and expression by this fusion was induced four- and 4·5-fold in the presence of 25 µM arsenite and arsenate, respectively (Fig. 4a).
Number and size of transcripts
To investigate whether there were two mRNA transcripts for the ars operon, and to estimate their sizes and whether they were regulated differently by arsenate and arsenite in E. coli, Northern-blot analysis was performed. Total RNA was prepared from E. coli ACSH50Iq(pTfarsCRBH-ACYC) cells. Probes for the arsR, arsB and arsHgenes and a region including the arsB and arsR genes (ars probe) were used (Fig. 1). About 10 µg of RNA was loaded into the wells of the gel shown in Fig. 5
. Attempts to obtain a stronger signal by loading 30 µg of RNA resulted in an increase in the signal from the degraded transcript smear and blot resolution was not improved. When RNA was isolated from At. ferrooxidans strains no transcripts were observed; hence, we believe that the ars genes are expressed at low levels in this organism.
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DISCUSSION |
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The ArsR regulators are part of the ArsR family of metalloregulatory proteins, which also includes CadC (the regulator of the Cd2+-efflux ATPase) and SmtB (the regulator of the metallothionein SmtA protein in response to the presence of zinc). In this study, we found that although ArsR from At. ferrooxidans does not contain the conserved metal-binding domain, there are many other strongly conserved areas within its sequence and the sequences of SmtB, other known ArsR proteins and its closest matches from the BLAST search. The crystal structure of SmtB has been determined and has been shown to have a winged helixturnhelix (HTH) structure (Cook et al., 1998 ). The secondary structure of SmtB consists of four
-helices and two ß-sheets, with the ß-sheets having been shown to form a hairpin structure. The conserved Gly-X-X motif (where X represents a large hydrophobic residue) at the end of the DNA-binding HTH domain is found in many of the proteins shown in Fig. 3(a)
. Cook et al. (1998)
believe that these hydrophobic residues are important for anchoring the ß-sheet that follows. Secondary-structure predictions indicate that many of these general features appear to be present in the second family of ArsR-like regulators. However, the absence of the established metal-binding motif and the observation that there are two extra amino-acid residues before the conserved hydrophobic region corresponding to the second helix of the HTH domain argue against a detailed extrapolation from the secondary structure of SmtB to the ArsR protein of At. ferrooxidans.
We have shown that at least two RNA transcripts, one corresponding to arsBH and the other corresponding to arsRC, were induced in the presence of arsenite and arsenate. Both Northern hybridization and reporter gene studies indicated that the level of expression from ParsB was low. Although expression was increased following induction, the levels of expression remained low. While this could be due to inefficient expression in E. coli, these low levels of expression were also observed when fusions to the ars genes from the Bacillus subtilis skin element were constructed and integrated into the chromosome (Sato & Kobayashi, 1998 ). The reason for such low levels of expression could be due to the toxic nature of the ars genes. Overexpression of the E. coli ArsB protein from a Ptachas been reported to be toxic to cells (e.g. Cai & DuBow, 1996
). Despite the low levels of expression, induction of expression by arsenite, arsenate and antimonite was observed.
The relative levels of expression from the arsRC promoter gave apparently conflicting results, depending on whether Northern hybridization or reporter gene studies were used. Although the quantity of arsRC transcript seen on the Northern blot was higher than for arsBH, the level of arsRClacZ expression was much lower than for the arsBlacZ fusion. The observation that the arsRC mRNA transcript was a smear may indicate that this mRNA has a higher turnover rate than arsBH mRNA. If much of the degradation takes place from the 5' end, this may be the reason for the lower level of reporter gene expression. Because reporter gene expression from the arsRC promoter was so low (less than 10 Miller units), we worked with a higher-copy-number vector and obtained results that were consistent with those observed in the low-copy-number vector. Expression from an arsRC promoter fused at the start of arsR was unresponsive to the presence of arsenic, presumably due to the absence of arsR. The addition of an arsR gene in trans did not induce expression, possibly because sequences downstream of the point of fusion were required or because the At. ferrooxidans arsR gene is not autoregulated. This inability to demonstrate autoregulation may indicate a difference between the two families of ArsR proteins and needs to be investigated further. However, when a translational fusion to arsC (which included arsR and the arsRC promoter) was investigated, expression was induced in the presence of both arsenite and arsenate. This indicated that arsR was required for regulation of this promoter. As an arsClacZ fusion containing the region upstream of arsC but not including the arsR promoter (pClacZ) showed no ß-galactosidase activity, it appears that arsR and arsCare expressed as a single transcript. Therefore, although the two ars transcripts of the At. ferrooxidans ars operon were transcribed at different levels, both were regulated by arsenite and arsenate.
We found that the last 19 aa of the C terminus of At. ferrooxidans ArsR were not required for either repression or induction of the arsBH promoter by arsenite, arsenate or antimony. However, when a further 28 aa of ArsR in ciswith the promoter fusion were deleted (leaving only 71 aa), expression from the promoter was constitutive. Similarly, it was shown that E. coli chromosomal ArsRß-lactamase chimeras were constitutive when fused to regions upstream of the 79th amino acid, but were inducible when ß-lactamase was fused downstream of the 92nd amino acid (Xu et al., 1996 ). These positions align with amino acids 85 and 98 of the At. ferrooxidans ArsR. An interesting observation is that the double cysteines conserved in At. ferrooxidans ArsR and its closest relatives were retained in the functional truncated ArsR. Further experiments are required to show whether these residues are involved in regulation.
We were unable to find a clear role for ArsH in the regulation of the At. ferrooxidans ars genes. While the addition of constructs containing arsH in trans with the arsB promoter fusions appeared to result in increased repression of the promoter and lower levels of induction, especially when arsenate was the inducer, these differences were small (Fig. 4b). We were unable to add the tac-regulated arsH gene back in trans, as we found that IPTG on its own decreased expression from the At. ferrooxidans arsB promoter. It is possible that the role that ArsH plays is only important in At. ferrooxidans and not in E. coli.
This is the first functional study of an ArsR regulator that is able to respond to arsenic, but which does not contain the conserved metal-binding motif. We propose that the ArsR protein from At. ferrooxidans is the first reported member of a subclass of ArsR regulators that may have a different method of binding the inducer to other ArsR regulators.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 10 April 2002;
revised 15 July 2002;
accepted 19 August 2002.