Characterization of a promiscuous cadmium and arsenic resistance mechanism in Thermus thermophilus HB27 and potential application of a novel bioreporter system
© The Author(s) 2018
Received: 12 February 2018
Accepted: 3 May 2018
Published: 18 May 2018
The characterization of the molecular determinants of metal resistance has potential biotechnological application in biosensing and bioremediation. In this context, the bacterium Thermus thermophilus HB27 is a metal tolerant thermophile containing a set of genes involved in arsenic resistance which, differently from other microbes, are not organized into a single operon. They encode the proteins: arsenate reductase, TtArsC, arsenic efflux membrane transporter, TtArsX, and transcriptional repressor, TtSmtB.
In this work we show that the arsenic efflux protein TtArsX and the arsenic responsive transcriptional repressor TtSmtB are required to provide resistance to cadmium. We analyzed the sensitivity to Cd(II) of mutants lacking TtArsX, finding that they are more sensitive to this metal than the wild type strain. In addition, using promoter probe reporter plasmids, we show that the transcription of TtarsX is also stimulated by the presence of Cd(II) in a TtSmtB-dependent way. Actually, a regulatory circuit composed of TtSmtB and a reporter gene expressed from the TtarsX promoter responds to variation in Cd(II), As(III) and As(V) concentrations.
Our results demonstrate that the system composed by TtSmtB and TtArsX is responsible for both the arsenic and cadmium resistance in T. thermophilus. The data also support the use of T. thermophilus as a suitable chassis for the design and development of As-Cd biosensors.
Toxic metals and metalloids such as cadmium (Cd) and arsenic (As) are widespread environmental contaminants that pose risks to human health . Microorganisms are endowed with multiple molecular mechanisms to handle exposure to these toxic compounds. In general, microbial resistance is achieved through three main mechanisms: transformation of the metals through reduction to a different oxidation state, efflux outside the cell by transporters, and/or sequestration/biosorption . Common reduction mechanisms include for example the conversion of Hg2+ to Hg0, AsO43− to AsO33−. Facilitated efflux transporters fall into two wide, functionally and evolutionary distinct membrane protein families, the P-type ATPases and the Major Facilitator Superfamily (MFS) of transporters [3–5]. P-type ATPases use ATP hydrolysis to transport ions across cellular membranes and are composed of three conserved domains: (1) the transmembrane (TM) helix bundle, allowing substrate translocation; (2) the soluble ATP binding domain (ATPBD) that contains the transiently phosphorylated Asp residue; (3) the soluble actuator domain (AD) . Over the years, on the basis of sequence similarity and overall architecture, they have been divided into different classes: those belonging to P1B-type are capable to drive the efflux out of cells of both essential transition metal ions (e.g., Zn2+, Cu+, and Co2+) and toxic metal ions (e.g., Ag+, Cd2+, Pb2+) contributing to their homeostasis maintenance. A recent study on a huge number of P1B-type ATPase sequences combined with available biochemical data classifies them into seven distinct subfamilies (1B-1 1B-7) on the basis of conserved motifs in TM4, TM5 and TM6, but the molecular basis of metal ion specificity remains unclear . All metal efflux transporters characterized so far are tightly controlled at transcriptional level by metalloregulatory proteins which bind DNA sequences and dissociate following metal binding, thus ensuring derepression of genes encoding efflux proteins . Several regulatory systems dedicated to metal/metalloid sensing have also been characterized; for instance, transcription factors of the ArsR/SmtB family are small dimeric proteins with a winged helix-turn-helix DNA binding domain controlling gene expression in response to divalent metals (e.g. zinc, cadmium) as well as metalloids (e.g. arsenic and antimonite) through an allosteric switching mechanism . The exploration of life in extreme environments has led to the isolation of many thermophilic microorganisms occupying diverse extreme habitats like hot hydrothermal fluids containing high concentrations of toxic metals. For this reason they are able to cope with toxic metals, which are more soluble at high temperatures, or even to use them for their metabolism [10, 11] and are currently exploited in some bioprocesses such as biomining and bioremediation . A detailed understanding of the molecular mechanisms responsible for resistance to toxic metals is also crucial for engineering organisms to develop sensitive biosensors for the detection of chemicals in the environment and to enhance bioremediation strategies [13, 14].
A significant number of whole-cell arsenic and/or cadmium biosensors has been already described in literature and is based on the realization of reporter systems containing regulatory cis-acting sequences interacting with a transcriptional repressor belonging to the ArsR/SmtB family [15, 16]. Biosensors are not intended to fully replace chemical methods but have the advantage of lower fabrication cost and higher stability and can offer on-site monitoring of even trace levels of targeted compounds in comparison with non-portable analytical methodologies . To date, the major challenges in biosensor development regard the screening or modification of efficient regulator protein/promoter pair for increased sensitivity and specificity [18, 19] as well as biosensor stability over time and simultaneous monitoring of multiple environmental parameters .
Thermus thermophilus HB27 is a thermophilic aerobic Gram-negative bacterium capable of growing in the presence of arsenic concentrations that are lethal for other microorganisms . In recent studies we demonstrated that the arsenic resistance system is not clustered in a single ars operon as in other organisms, but the genes are spread in the genome: TTC1502 encoding a cytoplasmic arsenate reductase (TtArsC) able to reduce arsenate to arsenite, TTC0354, encoding a P1B-type membrane ATPase responsible for the efflux (herein named TtArsX) and TTC0353 encoding a repressor (TtSmtB) sensitive to both As(V) and As(III) [22–24]. TtSmtB is a member of the ArsR/SmtB family, sharing 50% identity with the well characterized SmtB of Synechococcus PCC7942 . It is a dimeric protein containing three Cys residues in a reduced state and a conserved metal binding box presumably involved in As(V) and As(III) binding. The protein can bind to a consensus regulatory sequence located upstream of TtarsX preferentially in an un-metallated state and in vivo TtSmtB regulates TtarsX transcription upon arsenic interaction through a derepression mechanism .
In the present study, we evaluated the contribution of TtSmtB and TtArsX in cadmium detoxification using a combination of genetic and physiological approaches. The results obtained support that in T. thermophilus the mechanism employed for survival to cadmium and arsenic exposure is promiscuous, suggesting that the evolution of shared/common defense mechanisms represents an adaptation strategy to cope with toxic metals at high temperatures whereas keeping a reduced genome. In addition, to analyze the cadmium/arsenic response of TtarsX promoter in dependency on varying TtSmtB concentration, we settled up different β-gal reporter systems with the final goal of evaluating the utilization of thermophilic molecular components and thermostable chassis cells in biosensing applications.
Culture conditions and determination of minimum inhibitory concentration
Strains, genotypes and sources are summarized in Additional file 1: Table S1.
T. thermophilus HB27 wild type strain, T. thermophilus ΔsmtB::kat and TTC0354::pK18 mutants were grown aerobically at 70 °C in TM medium as described .
Minimum inhibitory concentration (MIC) was determined as the lowest concentration of cadmium that completely inhibited the visible growth of the strains after overnight incubation as indicated by the lack of turbidity. Basically, exponentially growing cultures of T. thermophilus HB27, T. thermophilus ΔsmtB::kat and TTC0354::pK18 were diluted to 0.08 OD600 nm in 24 well plates containing increasing concentrations of CdCl2 ranging from 0 mM to 5 mM and incubated at 70 °C or 60 °C for 18 h; depending on the strain tolerance, the concentration interval was narrowed in consecutive experiments; the MIC endpoint was considered as the lowest Cd(II) concentration at which there was a difference between grown and start culture lower than 0.01OD600 nm. The values reported are the average of two independent determinations.
In silico analysis
BlastP analyses were performed using the Blastall (v.2.2.25) program. The predicted presence and number of TMs, in the full length TtArsX, was determined using the TMHMM 2.0 online (http://www.cbs.dtu.dk/services/TMHMM) and the TM-pred online servers (http://www.ch.embnet.org/software/TMPRED_form.html) [26, 27]. Conserved motifs in TM helices of TtArsX were determined by manual inspection referring to those identified by Smith et al. . The presence of Metal Binding Domain(s) (MBD) was determined by manually looking at a CXXC motif in the protein sequence and fold prediction performed at (http://pfam.sanger.ac.uk).
Models of the MBD were generated using I-TASSER web server  (https://zhanglab.ccmb.med.umich.edu/I-TASSER/) using as input the first 91 amino acids of TtArsX. The model was selected according to its similarity to available crystal structures of MBD of P1B-type ATPases from other species .
The docking calculations were obtained using Hex Protein Docking server  with TtArsX MBD and As(III) or Cd(II); 100 rigid body docking solutions were generated and the 10 best refined by energy minimization. The proposed model for the metal docked into the MBD is the structure with the smallest distance between the metal and cysteine [2.61 Å for As(III) or 2.35 Å for Cd(II)], after an energy minimization step.
The regulatory region upstream of TtarsX (TtarsXp) previously named TTC0354p, was amplified by PCR using the primer pairs new 0354pr fwEcoRI and R0354NdeI, respectively (Additional file 1: Table S2); the region extends from − 73 to + 1 from the transcription start site . The primers introduced EcoRI and NdeI restriction sites, so that the amplified fragment could be cloned in the adapted pMHbgaA plasmid . The new vector was named pMHTtarsXpbgaA.
To obtain the plasmid pMHTtarsXpbgaA-nqoTtsmtB, the pnqo-TtsmtB gene cassette, where TtsmtB is under the control of the nqo promoter, was cloned into pMHTtarsXpbgaA vector; in T. thermophilus, the nqo promoter drives the expression of the operon encoding the major respiratory complex I during aerobic growth . In particular, pET28/TtsmtB was digested with NdeI HindIII  and cloned into the corresponding sites of pMKpnqo-bgaA  giving pMKpnqo-TtsmtB; afterwards, the plasmid was digested with XbaI HindIII and the gel purified pnqo-TtsmtB cassette subjected to a fill-in reaction and cloned into the filled-in HindIII site of pMHTtarsXpbgaA.
pMHTtarsXpbgaA-nqoTtSmtB was used to transform T. thermophilus HB27 and TTC0354::pK18 mutants in the conditions described . The pMHPnorbgaA vector was also used to transform the same strains and used as negative control . Cells were then incubated for 24–48 h at 60 °C on TM plates containing hygromycin (100 μg/mL). All the plasmids used in this study are described in Additional file 1: Table S3.
For measuring reporter β-galactosidase activity, the growing transformants were diluted to 0.1 OD600 nm in TM medium in the presence of hygromycin (100 μg/ml), treated with different concentrations of NaAsO2, KH2AsO4 and CdCl2 (Sigma) as source of As(III), As(V) and Cd(II) respectively, and grown at 60 °C for 16 h. β-galactosidase activity assays were carried out on permeabilized cells in 96-well microplates at 70 °C with a Synergy H4 microplate reader (BioTeK) as described by Miller .
Purification of TtSmtB
Recombinant TtSmtB was purified to homogeneity using the procedure already described, consisting of thermo-precipitation of the E. coli BL21-Codon Plus (DE3) RIL/TtSmtB cell extract followed by HiTrap Heparin chromatography. The histidine tag was removed from purified TtSmtB by thrombin digestion (Sigma). The purified protein was stored in aliquots at − 20 °C .
Electrophoretic mobility shift assay (EMSA)
To determine if cadmium was ligand of TtSmtB, electrophoretic mobility shift assays (EMSA) were performed. The TtarsX promoter region was amplified by PCR using specific primer pair: 0354footprint fw and 0354footprint rv, (Additional file 1: Table S2). EMSA reactions were set up as described , using 5 µM of proteins pre-incubated or not with Cd(II) at molar ratio of 1:20 and 1:50 (considering TtSmtB as a dimer).
Results and discussion
Domain organization and subfamily classification of TtArsX
Taken together, the results of in silico analysis assign TtArsX to the well characterized P1B-2-subfamily, show the presence of a soluble heavy metal associated domain (Pfam: PF 00403) and suggest a wider metal ion specificity than that previously known.
Contribution of TtArsX and TtSmtB to the Cd(II) tolerance mechanism
To analyse the role of TtArsX and the transcriptional repressor TtSmtB  in resistance to cadmium, we used different physiological and genetic approaches.
Bacterial resistance to cadmium and arsenic
T. thermophilus HB27
T. thermophilus TTC0354::pK18
T. thermophilus ΔsmtB::kat
In addition, the comparison of MIC values with those previously reported for As(V) and As(III) showed that T. thermophilus HB27 is almost 14-fold more sensitive to Cd(II) than to arsenic (Table 1).
Altogether, these results indicate that TtSmtB regulates cadmium tolerance by controlling at transcriptional level the metal efflux gene, adopting a derepression mechanism similar to that employed for arsenic detoxification .
Bioreporter construction and characterization
This work reports for the first time the identification of a molecular mechanism responsible for cadmium tolerance of the thermophilic bacterium T. thermophilus, taking advantage of the utilization of suitable genetic tools. Interestingly, the system includes part of the machinery (TtArsX and TtSmtB, membrane efflux ATPase and ArsR/SmtB transcriptional repressor, respectively) used to cope with arsenic; to the best of our knowledge this is the first functional characterization in a bacterium of a common/promiscuous mechanism to defend from both arsenic and cadmium, giving new venues for the understanding of the metal response evolution and the adaptation of environmental thermophilic microorganisms to deal with high concentrations of metals under enhanced solubilization conditions . Notably, from an evolutionary point of view it can be speculated that in the absence of an ars operon, cells may have evolved arsenic resistance from preexisting metal detoxification systems. It is also plausible that promiscuous detoxification systems have developed according to the hypothesis that in the genomes of thermophilic microorganisms the genetic information is condensed. Moving to biotechnologically relevant applications in the improvement of biosensor field, this study outlines the importance of a detailed characterization of the molecular components (intrinsic promoter activity, repressor/promoter and repressor/metal(s) binding affinities) and points to T. thermophilus as suitable chassis cell for design and development of robust metal biosensors. In this context, advantageous modifications can be programmed to increase biosensor sensitivity, selectivity and/or ability to detect metal mixtures.
IA, GG and ALR performed experiments. DL, PC, AB, JB, SB and GF supervised the project. IA and GF drafted the manuscript. All authors read and approved the final manuscript.
The authors thank Dr. Emilia Pedone of the CNR Institute of Biostructures and Bioimages in Napoli for helpful discussions during the preparation of the manuscript.
The authors declare that they have no competing interests.
Availability of data and materials
The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.
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This research was carried out in the frame of the Project “Immobilization of ENzymes on hydrophobin-functionalized NAnomaterials” funded by the University of Napoli Federico II.
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