OUP user menu

Characterization of a two-component signal transduction system that controls arsenite oxidation in the chemolithoautotroph NT-26

Sunita Sardiwal, Joanne M. Santini, Thomas H. Osborne, Snezana Djordjevic
DOI: http://dx.doi.org/10.1111/j.1574-6968.2010.02121.x 20-28 First published online: 1 December 2010


NT-26 is a chemolithoautotrophic arsenite oxidizer. Understanding the mechanisms of arsenite signalling, tolerance and oxidation by NT-26 will have significant implications for its use in bioremediation and arsenite sensing. We have identified the histidine kinase (AroS) and the cognate response regulator (AroR) involved in the arsenite-dependent transcriptional regulation of the arsenite oxidase aroBA operon. AroS contains a single periplasmic sensory domain that is linked through transmembrane helices to the HAMP domain that transmits the signal to the kinase core of the protein. AroR belongs to a family of AAA+ transcription regulators that interact with DNA through a helix-turn-helix domain. The presence of the AAA+ domain as well as the RNA polymerase σ54-interaction sequence motif suggests that this protein regulates transcription through interaction with RNA polymerase in a σ54-dependent fashion. The kinase core of AroS and the receiver domain of AroR were heterologously expressed and purified and their autophosphorylation and transphosphorylation activities were confirmed. Using site-directed mutagenesis, we have identified the phosphorylation sites on both proteins. Mutational analysis in NT-26 confirmed that both proteins are essential for arsenite oxidation and the AroS mutant affected growth with arsenite, also implicating it in the regulation of arsenite tolerance. Lastly, arsenite sensing does not appear to involve thiol chemistry.

  • histidine kinase
  • response regulator
  • AAA+ protein
  • arsenite sensing
  • arsenite oxidation


Arsenic is a naturally occurring toxic metalloid whose soluble forms, arsenite (H3AsO3) and arsenate (HAsO42−/H2AsO4−), can be used by certain prokaryotes for respiration (Stolz et al., 2006). Arsenite is most abundant in anoxic environments because, in oxic environments, it becomes readily oxidized to arsenate by arsenite-oxidizing bacteria –‘arsenite oxidizers’ (Stolz et al., 2006). Depending on their obligate source of carbon, arsenite oxidizers are either autotrophic or heterotrophic organisms that utilize either oxygen or nitrate as the terminal electron acceptor (Stolz et al., 2006).

Rhizobium sp. str. NT-26 is a facultative chemolithoautotrophic arsenite oxidizer that was isolated from the Granites goldmine, Northern Territory, Australia (Santini et al., 2000). It oxidizes arsenite using a periplasmic heterotetrameric arsenite oxidase (Aro), which is part of an electron transport chain involving a soluble c-type cytochrome and cytochrome oxidase (Santini & vanden Hoven, 2004; Santini et al., 2007). The arsenite oxidase gene cluster consists of four genes, aroB (encodes Rieske-like 2Fe–2S protein), aroA (encodes the catalytic subunit, which contains the molybdenum cofactor and a 3Fe–4S cluster), cytC (encodes a periplasmic cytochrome c552) and moeA1 (encodes a molybdenum biosynthesis gene), that are transcribed together when the organism is grown with arsenite. In the absence of arsenite, neither aroB nor aroA transcripts are detected even though a transcript for cytC and moeA1 is generated, suggesting that there are two separate transcriptional units under the control of two separate promoters (Santini et al., 2007). Only a single consensus sequence for a σ54-like promoter was located upstream of aroB (Santini et al., 2007).

The regulation of arsenite oxidase gene expression is poorly studied. In the closely related organism Agrobacterium tumefaciens str. 5A, which, unlike NT-26, cannot utilize arsenite as a source of energy, the genes in the homologous arsenite oxidase gene cluster [i.e. aoxA (=aroA), aoxB (=aroB) and cytC] are found within a single operon together with aoxR (encodes a putative transcriptional regulator) and aoxS (encodes a putative sensor histidine kinase) (Kashyap et al., 2006). The regulation of arsenite oxidation in A. tumefaciens is, however, complex such that it includes a quorum-sensing mechanism in addition to the putative two-component signal transduction system (AoxSR). In another heterotrophic arsenite-oxidizing bacterium, Ochrobactrum tritici SCII24, which also contains the arsenite oxidase gene cluster (i.e. aoxR, aoxS, aoxA, aoxB, cytC and moeA), the aoxR is transcribed separately from aoxA (Branco et al., 2009). Most recently, a differential transcriptome analysis was used to identify genes, in Herminiimonas arsenicoxydans that are involved in the response to arsenite (Koechler et al., 2010). Transposon insertions into aoxR and aoxS genes resulted in a lack of arsenite oxidase expression, thus demonstrating regulation of the aox operon by the AoxRS two-component system in this heterotrophic bacterium (Koechler et al., 2010).

In this report, we have identified and characterized two genes immediately upstream of the arsenite oxidase gene cluster in NT-26. We have also demonstrated that the two gene products designated AroS and AroR are essential for arsenite oxidation and comprise a classic two-component signal transduction pair that interacts through a phosphorelay reaction.

Materials and methods

Growth of NT-26

NT-26 was grown aerobically with shaking (130 r.p.m.) at 28 °C in a minimal salts medium (MSM) either chemolithoautotrophically with 5 mM arsenite or heterotrophically with 0.04% yeast extract with and without 5 mM arsenite. For growth experiments, cultures were grown for 18 h and inoculated (10% inoculum) into the experimental medium (100 mL). Samples were taken periodically and the OD600 nm was determined (Santini et al., 2000). Growth experiments were performed with two replicates on two separate occasions. For DNA isolations, NT-26 was grown as described previously (Santini & vanden Hoven, 2004). For RNA isolations, NT-26 was grown heterotrophically with and without arsenite until the mid log, late log and stationary phases. Arsenite oxidation was measured as reported previously (Santini et al., 2007).

Sequencing of aroR and aroS and production of heterologous expression constructs

DNA sequence upstream of the arsenite oxidase gene aroB was obtained by a primer walking method using a previously constructed genomic DNA library (Santini & vanden Hoven, 2004). To identify putative genes, the sequence results obtained were submitted to the database search engines smart (Schultz et al., 1998), pfam (Bateman et al., 2002) and tmhmm (Krogh et al., 2001). Sequence alignments were performed using either blastp (Camacho et al., 2008) or clustalw (Larkin et al., 2007). The aroR and aroS sequences have been deposited in GenBank under the accession number AY345225.

AroS and AroR genes were PCR amplified using genomic DNA (Santini & vanden Hoven, 2004) as a template. The digested amplified products were ligated into NcoI- and HindIII-digested pEMBL His-GST vector. Site-directed mutagenesis was performed using the QuikChangeTM Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA) protocol. All genes were sequenced (Eurofins MWG Operon) to verify cloning and to ensure that the correct mutations had been introduced. The constructs allowed for the overexpression of genes with an N-terminal polyhistidine affinity tag and a tobacco etch virus (TEV) protease cleavage site to allow for removal of the affinity tag.

Mutagenesis of aroR and aroS

Mutagenesis of aroR and aroS was performed by targeted gene disruption as described previously for aroA (Santini & vanden Hoven, 2004) and cytC (Santini et al., 2007). Portions of the aroR and aroS genes were amplified using the following primers: AroRFor (binds to nucleotides 31–50) 5′-GCGGATCCCTCGAAGATGATCCGATCAT-3′ (the recognition sequence for EcoR1 is underlined) and AroRRev (binds to nucleotides 709–728) 5′-GCGAATTCGCTGCATGACGCCAATCTCG-3′ (the recognition sequence for BamH1 is underlined); AroSFor (binds to nucleotides 222–242) 5′-GCGGATCCCTATGATCTGCTCGACCGTAC-3′ (the recognition sequence for EcoR1 is underlined) and AroSRev (binds to nucleotides 1082–1102) 5′-GCGAATTCTGCTCATGCACGTCAATGTCT-3′ (the recognition sequence for BamH1 is underlined). The PCR products were digested with EcoR1 and BamH1 and cloned into the suicide plasmid pJP5603 (KmR) and transferred into NT-26 by conjugation (Santini & vanden Hoven, 2004; Santini et al., 2007). One aroR and one aroS mutant were chosen for further study.

Mutants were tested for their abilities to grow chemolithoautotrophically and heterotrophically. As no growth was detected with either mutant when grown chemolithoautotrophically with 5 mM arsenite, growth experiments were only conducted under heterotrophic conditions. Growth experiments were conducted with two replicates on two separate occasions in batch cultures in the MSM with 0.04% yeast extract with and without 5 mM arsenite.

Expression analysis of aroR and aroS in NT-26

Reverse transcriptase (RT)-PCR was used to assess the transcription of the aroR and aroS genes. NT-26 was grown heterotrophically with 0.04% yeast extract with and without 5 mM arsenite. Cells were harvested at three different growth phases, namely the mid log (OD600 nm 0.098), late log (OD600 nm 0.036) and stationary (OD600 nm 0.14) phases. RNA isolation and RT-PCR were performed as described previously (Santini et al., 2007). The primers used to detect the expression of aroS and aroR, respectively, were as outlined above for the targeted gene disruption. PCR product sizes were 880 bp for the sensor kinase gene and 697 bp for the regulatory gene. The primers used to detect expression of aroB were as described previously (Santini et al., 2007).

Heterologous protein expression and purification

Overexpression of all genes was carried out in Escherichia coli Rosetta (DE3) pLysS cells. Protein expression was induced by the addition of 0.5 mM IPTG and the culture was allowed to grow for a further 12-h shaking at 18 °C. The cells were then harvested by centrifugation and the pellets were stored at −20 °C until required. The cells were defrosted on ice and resuspended in buffer A [25 mM Tris, 200 mM NaCl, pH 8.5, complete EDTA-free cocktail inhibitor (Roche)] and then lysed by sonication (10 bursts of 30 s each with 1-min interval). The lysate was centrifuged at 13 000 g for 1 h. The supernatant was incubated with Ni-NTA agarose (Qiagen) with agitation for 1 h. After incubation, the beads were washed four times with 15 bead volumes of buffer A containing 20 mM imidazole. The protein was eluted in buffer A containing 250 mM imidazole and the eluted fraction was checked by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). TEV protease was added in a 1 : 10 dilution of the total amount of protein present, and the solution was left to dialyse overnight at 4 °C in 50 mM Tris-HCl, pH 8.0, 200 mM NaCl and 2 mM β-mercaptoethanol. To remove the cleaved protein tag and TEV protease, the dialysed solution was passed over Ni-NTA agarose (Qiagen), and the unbound cleaved protein was collected.

In vitro phosphorylation assays

The recombinant protein AroS226–490 (15 μM) was assayed for the ability to autophosphorylate in a reaction mixture containing 5 μCi [γ-32P]ATP (NEN Radiochemicals), 100 mM Tris-HCl, pH 8.0, 10 mM MgCl2 and 50 mM KCl in a final volume of 100 μL. The reaction was incubated at room temperature for 15 min; 20 μL of the reaction sample was removed at 1-, 5-, 10- and 15-min intervals and quenched with the addition of 5 μL of a stop buffer solution consisting of 250 mM Tris pH 6.8, 10% glycerol, 1% SDS, 280 mM β-mercaptoethanol and 0.01% bromophenol blue. Phosphotransfer was assayed such that 10-μL aliquots of AroS226–490, which was first autophosphorylated for 10 min, were combined with 15 μM purified AroR1–125 or AroR1–125D13N or AroR1–125D53N or AroR1–125D58N protein. Reaction mixtures were incubated simultaneously at room temperature for the indicated time periods. AroS226–490 H273N and AroS226–490 H292N were additionally analysed for their ability to autophosphorylate in the same manner as AroS226–490, with the exception of only taking a 10-min incubation time sample. In all the phosphorylation assays, samples were analysed by SDS-PAGE and autoradiography overnight.

1D nuclear magnetic resonance (NMR) spectroscopy

All 1D 1H NMR spectra were recorded at a 1H frequency of 700 MHz on a Bruker Advance III spectrometer at 25 °C in a buffer containing 20 mM sodium phosphate, pH 8.0, and 150 mM NaCl using protein samples at 0.1 mM concentration.


Two genes, aroS and aroR, coding for putative histidine kinase and a response regulator are located upstream of the arsenite oxidase gene cluster

Bioinformatic analysis of a DNA sequence upstream of the arsenite oxidase gene aroB allowed for the identification of two ORFs (Fig. 1a). The first ORF, designated aroR, contains 1323 base pairs encoding a putative protein of 441 amino acids; the second ORF, named aroS, contains 1470 base pairs encoding a putative protein of 490 amino acids. Analysis of AroS and AroR amino acid sequences revealed their similarity to a typical two-component system signalling protein, where aroS codes for a sensor histidine kinase while aroR codes for a response regulator (Fig. 1b). The AroS protein is characterized by the presence of a dimerization and histidine phosphotransfer domain (DHp; residues 263–329) and an ATP-binding catalytic domain (CA; residues 370–480) in its C-terminus (Fig. 1b); the two domains are commonly found in a classical input component of a two-component signalling pathway. The DHp domain contains a conserved histidine residue that undergoes ATP-dependent phosphorylation, through the activity of the CA domain, in response to changes in the external environment. Sequence alignments identified the histidine residue located at position 273 as the presumed site of autophosphylation (Fig. 2a). In addition, AroS is predicted to contain two transmembrane segments within its N-terminus. Transmembrane segment 1 is proposed to include residues 14 through 32, while transmembrane segment 2 lies between residues 175 and 194. Present between these two transmembrane segments is the environmental stimuli-sensing portion of the protein, the sensory domain. Sequence analysis of this domain revealed that although the NT-26 AroS protein shares significant sequence identity with sensory domains from soil bacteria A. tumefaciens (80%) and O. tritici (79%), no significant homologue of a known structure could be identified. However, the length of the domain, secondary structure prediction and a weak homology to other unrelated sensory proteins would suggest that the regions fold most likely into a PAS-like topology. Interestingly, no cysteine residues are present in NT-26 AroS, implying that arsenite sensing and binding does not involve thiolate, as it is the case in other known arsenite-binding proteins (Mizumura et al., 2010). In contrast the AroS homologue in A. tumefaciens does contain a Cys at position 401, which has been implicated in binding arsenite (Kashyap et al., 2006).

Figure 1

(a) Organization of arsenite oxidase gene clusters from published arsenite oxidizers. NT-26; Agrobacterium tumefaciens str. 5A (DQ151549); Ochrobactrum tritici str. SCII24 (FJ465505); Alcaligenes faecalis (AY297781); Achromobacter sp. str. SY8 (EF523515); and Herminiimonas arsenicoxydans str. ULPAs1 (NC_009138). aroS, sensor histidine kinase gene; aroR, transcriptional regulator gene; aroA, arsenite oxidase large subunit gene; aroB, arsenite oxidase small subunit gene; cytC, cytochrome c gene; moeA, molybdenum cofactor biosynthesis gene (Note: The gene sequence of A. tumefaciens str. 5A moeA is partial), oxyX, oxyanion-binding gene; pho, phosphate/phosphonate ABC transporter. (b) Schematic representation of the putative domains and domain boundaries predicted for AroS sensor and AroR regulator. Predictions were obtained using the databases smart (Schultz et al., 1998), pfam (Bateman et al., 2002) and tmhmm (Krogh et al., 2001). TM, transmembrane region, HAMP, histidine kinases, adenylyl cyclases, methyl-binding proteins and phosphatases; CA, ATP-binding domain; DHp, histidine kinase phosphotransfer and dimerization domain; REC, receiver domain; AAA, ATPases associated with a variety of cellular activities; HTH_8, helix-turn-helix DNA-binding motif.

Figure 2

(a) Multiple sequence alignment of the DHp domain of AroS. The NCBI database was searched with blastp (Camacho et al., 2008) using AroS263–329 as a query. The top 10 results were retrieved and aligned using clustalx (Larkin et al., 2007). #A putative phosphorylated histidine residue. The number of amino acids between the MET residue and the start of the DHp domain are indicated. The number of the last amino acid in the alignment is given, followed by the overall protein length in parentheses. GenBank accession numbers are given, followed by abbreviations for the organism: Aeh, Alkalilmnicola ehrlichii str. MLHE-1 (GenBank accession number ACS43235); Mex, Methylobacterium extroquens str. AM1 (ABI55574); Afa, Alcaligenes faecalis (AAQ19841); Otr, Ochrobactrum tritici (ACK38264); Atu, Agrobacterium tumefaciens str. 5A (ABB51924); Ach, Achromobacter sp. str. SY8 (ACJ83256); Xau, Xanthobacter autotrophicus str. Py2 (ABS69171); Ros, Roseovarius sp. str. 217; Rfe1 and Rfe2, Rhodoferrax ferrireducens str. T118 (ABD70797 and ABD70806). (b) Multiple sequence alignment of the REC domain of AroR. The NCBI database was searched with blastp using AroR6–118. The top 10 results were retrieved and aligned using clustalx. #A putative ASP residue involved in transphosphorylation. The number of amino acids between the MET residue and the start of the REC domain are indicated. The number of the last amino acid in each row is given and the overall protein length in parentheses. GenBank accession numbers are given, followed by abbreviations for the organism: Aeh, Alkalilmnicola ehrlichii str. MLHE-1; Afa, Alcaligenes faecalis; Ros, Roseovarius sp. str. 217; Tha, Thauera sp. str. MZ1T; Otr, Ochrobactrum tritici; Atu, Agrobacterium tumefaciens str. 5A; Xau, Xanthobacter autotrophicus str. Py2; Rho, Rhodobacter sp. str. SW2; Dar, Dechloromonas aromatica str. RCB; Gfe, Gallionella ferrugenia str. ES-2. The following symbols denote the degree of conservation observed in each column: ⋆, the residues are identical in all sequences; :, conserved substitutions;., semi-conserved substitutions.

Sequence analysis of AroR identified a canonical two-component response regulator receiver domain (residues 6–118) in the N-terminal region of the protein sequence (Fig. 1b). The receiver domain, a signature structural feature in response regulator proteins, is subjected to phosphorylation activation mechanisms on a specific aspartate residue to initiate a response. There are four conserved aspartate residues within the amino acid sequence of AroR (Fig. 2b), with aspartate 58 residue predicted to be the most likely site of transphosphorylation. The receiver domain is linked to an AAA+ATPase domain that precedes the DNA-binding domain at the C-terminus. The presence of an AAA+ATPase domain is indicative of a transcription factor activity associated with the activation of σ54 promoters. In analogous response regulators from the NtrC/DctD family, ATPase activity is coupled to a hexameric or a heptameric ring assembly that is required for the formation of an open RNA polymerase complex at the initiation of transcription (Gao & Stock, 2009). Furthermore, AroR sequence analysis shows the presence of a highly conserved ESELFGHEKGAFTGA sequence motif that is essential for binding to the σ-factor of the σ54-RNAP (Yan & Kustu, 1999; Xu & Hoover, 2001; Bordes et al., 2003). We have previously detected a putative σ54-like promoter region upstream of aroB, and in a recent study of H. arsenicoxydans, it was shown, through transposon insertions, that alternative N sigma factor (σ54) of RNA polymerase is involved in the control of the arsenite oxidase gene expression (Koechler et al., 2010).

aroR- and aroS-like genes appear to be conserved within gene clusters associated with arsenite oxidation (Fig. 1a). However, in members of the Alphaproteobacteria that include NT-26, the aroR and aroS genes are in the same orientation as the arsenite oxidase genes, whereas in members of the Betaproteobacteria, they are in the opposite orientation with a gene involved in oxyanion binding or phosphate/phosphonate transport in between them (Fig. 1a). Both AroS and AroR share high sequence similarity (∼80% identity) to analogous proteins from A. tumefaciens and O. tritici, with sequence similarities declining significantly to the next closest sequence homologues from Xanthobacter autotrophicus exhibiting sequence identities of 43% and 56% for AroS and AroR, respectively. In all the other identified organisms, which have homologous proteins, sequence identities range from approximately 38% to 23% for AroS-like proteins and 43% to 38% for AroR-like proteins, with significantly higher sequence conservation of AroR compared with that of AroS, possibly reflecting differences between various stimuli activating these sensors.

Expression of both aroR and aroS is necessary for arsenite oxidation

The arsenite oxidase gene cluster consisting of aroB, aroA, cytC and moeA1 encodes two transcripts, one transcript that is constitutive and only contains cytC and moeA1 and another transcript that is inducible with arsenite and that contains all the genes and that is most likely regulated through an involvement of a putative σ54-like promoter upstream of aroB (Santini et al., 2007). To determine patterns of expression for the aroR and aroS genes, RT-PCR experiments were carried out with RNA isolated at different growth phases. The expression of both aroS and aroR was found to be constitutive as it did not require growth with arsenite (Fig. 3, lanes 2–3) and an aroS/aroR transcript was found to be in a separate transcriptional unit to aroB – an arsenite-induced gene (Fig. 3, lane 4).

Figure 3

RT-PCR analysis of the arsenite oxidase regulatory region. Sizes of markers are shown (lane 1). Lane 2 shows the amplification of a portion of aroS. Lane 3 shows the amplification of a portion of aroR. Lane 4 shows no product when an attempt is made to coamplify aroR and aroB.

The role of both AroR and AroS in arsenite oxidation was assessed through mutating each gene by targeted gene disruption (Santini & vanden Hoven, 2004; Santini et al., 2007) and then testing the ability of mutant strains to grow and oxidize arsenite both chemolithoautotrophically and heterotrophically. Neither aroR nor aroS transcripts could be detected in the aroS mutant, suggesting that a mutation in aroS has a downstream effect on the transcription of aroR (data not shown); a downstream effect on the transcription of aroB and aroA is not expected as these genes are transcribed in a different operon. A summary of the growth experiments is presented in Table 1. Both mutants were unable to oxidize arsenite under any conditions, with RT-PCR experiments showing that in both cases, arsenite oxidase gene aroB was not transcribed, while the expression of a downstream cytC gene, which belongs to a separate transcriptional unit (Santini et al., 2007), was not affected by the mutations. In addition, no cell growth was detected under chemolithoautotrophic conditions with 5 mM arsenite as the electron donor for either of the mutants. No effect on growth was observed when both mutants were grown heterotrophically with yeast extract (0.04%) alone with generation times of 2.6 h for the wild type and the AroS mutant, and 2.7 h for the AroR mutant. However, when the cells were grown heterotrophically with 0.04% yeast extract and 5 mM arsenite, the growth rate of the AroS mutant was significantly affected; the generation time of the wild type and the AroR mutant was 2.8 h, while the AroS mutant had a generation time of 3.8 h. These results show that both AroR and AroS are required for arsenite oxidation by providing transcriptional regulation of the arsenite-inducible arsenite oxidase (aroBA) transcript. In addition, AroS may play a role in the regulation of another pathway possibly involved in tolerance to arsenic, as the growth of the AroS mutant in arsenite-containing medium was slower than when the cells were grown with yeast extract alone. The role of AroS in arsenite tolerance will be further explored.

View this table:
Table 1

Effect of mutations in aroS and aroR on NT-26 cell growth

StrainGrowth conditions
5 mM arsenite0.04% (w/v) yeast extract0.04% (w/v) yeast extract+5 mM arsenite
ΔaroSND2.6 ± 0.013.8 ± 0.05
ΔaroRND2.7 ± 0.012.8 ± 0.05
WT7.62.6 ± 0.012.8 ± 0.05
  • The numbers in the column refer to the generation time in hours.

  • * Santini (2000).

  • ND, not detected.

AroS histidine kinase activity

The full-length AroS protein as well as the gene construct coding for the core kinase region (residues 226–490) were expressed in, and purified from, E. coli. The recombinant full-length AroS protein appeared insoluble, presumably due to the presence of the two transmembrane domains. Protein activity was therefore tested using the AroS226–490 protein fragment, containing the DHp domain and the CA domain (Fig. 1b), which was purified from the soluble fraction of the E. coli cell extracts. To study the ability of AroS226–490 to undergo autophosphorylation in vitro, the protein was incubated in the presence of [γ-32P]ATP and the time course of the reaction was followed (Fig. 4). AroS was readily phosphorylated, with the maximum incorporation of [γ-32P]ATP reached within 5 min as shown by the intensities of the bands in the auotoradiograph (Fig. 4a). Identification of the putative phosphoacceptor residue was carried out by site-directed mutagenesis of the only two histidine residues present in the phosphotransfer domain (DHp): His273 and His292. While the autophosphorylation activity of the AroS226–490H292N mutant was unaffected compared with the wild-type protein (Fig. 4c, lanes 2 and 3, respectively), the AroS226–490H273N mutant protein was defective in autophosphorylation (Fig. 4c, lane 1). Similar protein concentrations were used in these experiments as can be seen in Fig. 4b and d. Thus, we demonstrated that AroS exhibits sensor histidine kinase activity and that His273 is required for autophosphorylation most likely as the phosphoaccepting residue. 1D 1H NMR spectra of AroS226–490, AroS226–490H273N and AroS226–490H292N mutant proteins, recorded on a 1H frequency of 700 MHz on a Bruker Advance III spectrometer at 25 °C, were similar (see Supporting Information, Fig. S1), exhibiting characteristic features of a folded polypeptide, thus excluding the possibility that the loss of autophosphorylation of AroS226–490H273N is due to protein missfolding.

Figure 4

Autophosphorylation of AroS. (a) An autoradiograph of a time-dependent (min) autophosphorylation of AroS226–489. (b) Fifteen per cent SDS-PAGE gel of the same reaction. Lane C in (a) and (b) contains a control reaction with no [γ-32P]ATP added. Lane M contains molecular weight markers. (c) An autoradiograph confirming that His273 is the site of autophosphorylation in AroS226–489 (d) 15% SDS-PAGE gel of the same reaction: 1, AroS226–489 H273N+[γ-32P]ATP; 2, AroS226–489H292N+[γ-32P]ATP; 3, AroS226–489+[γ-32P]ATP; the reactions were stopped after 10 min of incubation.

AroS and AroR are cognate histidine kinase – response regulator pair

To address whether AroR is the cognate response regulator for AroS, an expression construct coding for the receiver domain of AroR (residues 1–125) was cloned and expressed in E. coli and recombinant protein AroR1–125 was purified. The transphosphorylation reaction was carried out such that AroS226–490 was first incubated with [γ-32P]ATP for 10 min to generate a population of phosphorylated AroS226–490 and then purified AroR1–125 was added to the reaction mixture. The transphosphorylation reaction of AroS226–490 with AroR1–125 was incubated at room temperature for 1 and 10 min. Figure 5 clearly shows the autophosphorylation of AroS226–490 and the subsequent transfer of the phosphate group to AroR1–125 (Fig. 5a, lanes 3 and 4). Phosphorylation of AroR1–125 is AroS-dependent as omission of AroS226–490 from the reaction mixture (Fig. 5a, lane 2 and c, lane 2) leads to no AroR phosphorylation – an expected observation, given that the receiver domains are unable to undergo ATP-dependent autophosphorylation. Direct phosphotransfer from AroS to AroR confirms that these two proteins are a cognate sensor response regulator pair.

Figure 5

In vitro phosphorelay reaction between AroS226–489 and AroR1–125. (a) Autoradiograph and (c) 15% SDS-PAGE gel: 1, AroS226–489+[γ-32P]ATP (10 min); 2, AroR1–125+[γ-32P]ATP (10 min); 3, AroS226–489+AroR1–125+[γ-32P]ATP (1 min); 4, AroS226–489+AroR1–125+[γ-32P]ATP (10 min). The site of transphosphorylation in AroR1–125 was confirmed by mutagenesis. (b) Autoradiograph and (d) 15% SDS-PAGE gel: 1, AroS226–489+AroR1–125+[γ-32P]ATP (1 min); 2, AroS226–489+AroR1–125+[γ-32P]ATP (10 min); 3, AroS226–489+AroR1–125D13N+[γ-32P]ATP (1 min); 4, AroS226–489+AroR1–125D13N+[γ-32P]ATP (10 min); 5, AroS226–489+AroR1–125D53N+[γ-32P]ATP (1 min); 6, AroS226–489+AroR1–125D53N+[γ-32P]ATP (10 min); 7, AroS226–489+AroR1–125D58N+[γ-32P]ATP (1 min); 8, AroS226–489+AroR1–125D58N+[γ-32P]ATP (10 min); 9, AroS226–489+[γ-32P]ATP (10 min). Smaller amounts of AroR1–125D53N were used in the reactions reflecting lower expression levels of this protein. Even though the protein level was lower than that of the D58N mutant, significant levels of transphosphorylation were detected.

To determine which aspartate residue is involved in the phosphorelay mechanism, purified protein variants of AroR1–125 containing single mutations (D13N, D53N and D58N) were tested for their ability to undergo transphosphorylation. Figure 5b shows that both AroR1–125D13N and AroR1–125D53N mutants show a reduced phosphorylation level (Fig. 5b, lanes 3–6) compared with wild-type AroR1–125 (Fig. 5b, lanes 1 and 2) and that there was no detectable phosphorylation of the AroR1–125D58N mutant protein (Fig. 5b, lanes 7 and 8). The canonical three-dimensional structure of the receiver domain contains an ‘acidic pocket’ that is essential for phosphorylation of the response regulator, although only one of the aspartate residues is ultimately phosphorylated. Our results suggest that Asp58 is the conserved transphosphorylation site in AroR that, together with Asp13 and Asp53, forms the acidic pocket. Again, we used 1D 1H NMR spectroscopy to confirm that the protein products used in these experiments were correctly folded.


Arsenite-oxidizing bacteria were first identified in 1918 (Green, 1918); however, until the last decade, none were found that utilized arsenite as an energy source (Santini et al., 2000; Stolz et al., 2006). We have now demonstrated that in the chemolithoautotroph NT-26, the specific two-component signal transduction system is involved in the transcriptional regulation of the arsenite-oxidizing enzyme. While previously putative regulatory genes have been reported from other arsenite-oxidizing organisms, we have for the first time demonstrated the enzymatic activities of the gene products and confirmed the two proteins as a cognate response regulator pair.

The main aspect of the regulation of arsenite oxidation is that it involves σ54-dependent transcription as indicated by the presence of a σ54 promoter region upstream of aroB and the identification of an AAA+ protein domain, which has been linked to σ54 activation in other systems, in the response regulator AroR. Approximately 10% of all known DNA-binding response regulators contains the NtrC/DctD AAA+ATPase domain fused to a factor of an inversion (Fis)-type helix-turn-helix domain (Batchelor et al., 2008; Gao & Stock, 2009). ATPase in the AAA+ proteins is dependent on the formation of a hexameric or a heptameric ring structure that is regulated by phosphorylation of the receiver domain (Gao & Stock, 2009). Currently, there are two known modes of phosphorylation-induced assemblies: in the case of NtrC phosphorylated REC domain participates in the intermolecular interactions and is involved in the formation of a hexameric interface (Kostrewa et al., 1992; Sallai & Tucker, 2005; De Carlo et al., 2006), whereas in the case of NtrC1 and DctD REC phosphorylation releases the inhibitory affect that this domain has on the formation of heptameric ring and ATPase activation (Park et al., 2002; Lee et al., 2003). Further structural and mechanistic studies will be carried out addressing the molecular basis and phosphorylation dependence of AroR–DNA interaction.

Arsenite sensing is particularly interesting from the aspect of bioremediation as arsenic contamination is a serious world-wide problem. In Asia (e.g. Bangladesh, several states of India, Nepal, Pakistan, Vietnam, Cambodia, China, etc.), more than 100 million people are estimated to be at risk, and some 700 000 people are known so far to have been affected by arsenic-related diseases (Rahman et al., 2009). Thus, the question of arsenite binding is of great interest to synthetic biologists involved in engineering of novel molecular entities that could be used in arsenite detection and decontamination. Most of the molecular models of arsenite binding involve thiol-based chemistry. In fact, most of the proteins that have been identified to bind to arsenite and thus have been inactivated by it do so through Cys residues (Hughes, 2002; Kitchin & Wallace, 2004). However, neither Cys residue nor Tyr residues, which have also been reported to bind arsenite in some proteins (Page & Wilson, 1985), are present within the sensory domain of AroS. Perhaps the difference in the mode of binding arsenite is not too surprising when considering the function that AroS performs. This protein needs to be able to bind arsenite reversibly and to be able to respond to changes in arsenite concentrations. Presently, we cannot provide a definitive answer of what the mechanism of arsenite sensing is; however, our work provides a foundation for further structural and mechanistic analysis of this regulatory system.

In addition, not only do arsenite-oxidizing bacteria need to be able to sense the presence of arsenite in the environment, but they also need means of evading arsenite toxicity. Our studies have demonstrated for the first time that a mutation in aroS has an effect on the growth of NT-26 in the presence of arsenite. Thus, AroS may play an additional role in the regulation of a pathway involved in tolerance to arsenite.

Supporting Information

Additional Supporting Information may be found in the online version of this article:

Fig. S1. 1D 1H NMR spectra for AroS226–490H273N protein that lacks autophosphorylation activity.

Please note: Wiley-Blackwell is not responsible for the content or functionality of any supporting materials supplied by the authors. Any queries (other than missing material) should be directed to the corresponding author for the article.


T.H.O. is supported by a Natural Environment Research Council studentship (14404A).


  • Editor: J. Colin Murrell


View Abstract