ARTICLE Selective Detection of Gold Using Genetically Engineered Bacterial Reporters

Sebastian Cerminati, Fernando C. Soncini, Susana K. Checa Facultad de Ciencias Bioqumicas y Farmaceuticas, Departamento de Microbiologa, Universidad Nacional de Rosario, Instituto de Biologa Molecular y Celular de Rosario, Consejo Nacional de Investigaciones Cientcas y Tecnicas, Suipacha 531, S2002LRK-Rosario, Argentina; telephone: 54-341-4356369; fax: 54-341-4390465; e-mail: checa@ibr.gov.ar
Received 11 March 2011; revision received 20 April 2011; accepted 13 May 2011 Published online 26 May 2011 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/bit.23213

ABSTRACT: Salmonella typhimurium harbours a Au-resistance system whose expression is controlled by GolS, a transcriptional regulator of the MerR family that selectively detects Au with high sensitivity. We developed both Salmonella and genetically engineered Escherichia coli strains as Au-selective whole-cell biosensors by coupling the strictly regulated GolS-dependent golB promoter to the gfp reporter gene. The bio-reporters were evaluated under different laboratory conditions and calibrated for their use as selective Au detectors. Due to the intrinsic characteristics of the regulatory protein, the transgenic E. coli sensor exhibits low background, high signal-to-noise ratio, and improved sensitivity for detection of Au ions in a wide range of concentrations (up to 470 nM) with a calculated detection limit of $33 nM (6 mg L1 or parts per billion) Au(I). The uorescent Au-sensing bacteria exhibit also minimal interference by chemically related metals such as Cu or Ag that are commonly found in Au deposits. These highly specic and sensitive Au detectors might allow the development of rapid and robust screening tools to improve discovery and extraction procedures. Biotechnol. Bioeng. 2011;108: 25532560. 2011 Wiley Periodicals, Inc. KEYWORDS: whole-cell bacterial biosensor; selective gold detection; Au-induced uorescent reporter; Salmonella GolS; transgenic golTSB E. coli strain; limits of Au detection

Introduction
Genetically engineered microorganisms emerge as attractive alternatives to conventional analytical methods for detection
Correspondence to: S.K. Checa Contract grant sponsor: Agencia Nacional de Promocion Cientca y Tecnologica Contract grant sponsor: National Research Council (CONICET) Additional supporting information may be found in the online version of this article.

of toxic compounds in environmental samples (Robbens et al., 2010; van der Meer and Belkin, 2010; Wanekaya et al., 2008). These biosensors harbor a sensing/response unit (in the simplest case, a transcriptional regulator and its controlled promoter) linked to a promoterless reporter gene whose product provides a measurable signal. The most commonly employed reporter genes are those coding for the marine jellysh Aequorea victoria green uorescent protein (gfp), and the genetic determinants of light production from the photoluminescent marine bacterium Vibrio sheri (lux) or in rey (luc) (Harms, 2007; van der Meer and Belkin, 2010). In recent years, engineered reporter bacteria have been developed for detection of several heavy metals and metalloids, including Hg, Cr, As, Cd, Pb, Ni, Co, Zn, and Cu (Harms, 2007; Hynninen and Virta, 2010; Magrisso et al., 2008; and references there in). In these biosensors, the metal-responsive regulatory unit originates from bacterial species that are naturally resistant to a particular heavy metal and usually depends on MerR- or ArsR/SmtB-like regulators. In MerR-like based biosensors, the metal ion interacts with the regulator which is bound to its target DNA sequence and induces a conformational change in the protein/DNA complex that promotes transcription of the reporter gene (Brown et al., 2003; Ma et al., 2009). Sensitivity and specicity of the response depend mainly on the inherent binding characteristics of the regulatory unit and the ability of the sensor protein to discriminate between similar signals. In fact, most of the designed MerR-based biosensors display high sensitivity but low selectivity because the sensor proteins cross-react with a group of related metal ions (Harms, 2007; Hynninen and Virta, 2010; Magrisso et al., 2008). For example, previously constructed Cu biosensors based on CueR, the monovalent metal sensor of the MerR family, detect Cu but also Ag and Au, as the regulator is similarly sensitive to any of these metals (Charrier et al., 2011; Hakkila et al., 2004; Riether et al.,

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2001; Stoyanov and Brown, 2003). This makes the use of these bioreporters unsuitable for particular applications. Gold is continuously demanded both as a valuable commodity and by modern industry because of its unique physicochemical properties that make it appropriate for many applications. Thus, efforts have been directed to improve both the discovery of economically suitable gold ores and the extraction procedures (Butt and Hough, 2009). Several analytical methods, such as ame atomic absorption spectrometry (FAAS), inductively coupled plasma optical emission spectrometry (ICP-OES), ICP mass spectrometry (ICP-MS), and the expensive neutron activation analysis (NAA), have been used to evaluate Au content in deposits (Caporali et al., 2010; Pitcairn et al., 2006). All these methods are sensitive enough to detect total Au content in soils and alluvial deposits, but require sophisticated instrumentation and trained personnel. In addition, some of them are laborious and time-consuming. Whole-cell bacterial biosensors have the potential to become simpler, cost-effective alternative methods to the conventional detection procedures (Harms, 2007; Hynninen and Virta, 2010; Magrisso et al., 2008). The Cu-responsive bacterial sensors based on CueR can be used to detect Au, because the transcriptional regulator binds Cu, Ag, and Au ions with similar afnity (Changela et al., 2003; Stoyanov and Brown, 2003). However, because of cross-detection their use is not recommended for analyzing Au content in minerals where the metal is usually combined with silver and copper (Hough et al., 2009). In this study, we developed whole-cell bacterial sensors that selectively detect Au. These sensors are based on the
Table I. Bacterial strains and plasmids used in this study.

regulatory unit controlling resistance to this toxic metal in Salmonella enterica serovar typhimurium (S. typhimurium) (Checa et al., 2007; Pontel et al., 2007). In the bacterial sensor, expression of the gfp reporter gene is directed by the Salmonella golB promoter which is transcriptionally controlled by GolS. The transcriptional regulator is homologous to CueR, but in contrast to the Cu-sensor, GolS distinguishes Au from either Cu or Ag ions (Checa et al., 2007). With the aim of using it as a biotechnological tool, we introduced the sensor/regulatory gene in the chromosome of a non-pathogenic E. coli strain that has been widely used in the design of whole-cell sensors (Robbens et al., 2010). The designed bacterial bioreporter displays good sensitivity and selectivity to quantify Au in complex samples from deposits.

Materials and Methods

Bacterial Strains and Growth Conditions S. enterica and Escherichia coli strains and plasmids used are listed in Table I. Details on the construction of bacterial strains and plasmids are provided as Supplementary Material. Bacterial strains were stored as frozen glycerol (15%) stocks at 708C. Bacteria were routinely grown at 378C in LuriaBertani (LB) broth or LB-agar plates, except for metal-induction assays. Kanamycin was used at a nal concentration of 50 mg mL1, tetracycline at 12.5 mg mL1, chloramphenicol at 20 mg mL1, and streptomycin at 100 mg mL1. All reagents, chemicals, and oligonucleotides were from Sigma-Aldrich Co.

Relevant properties Strain S. typhimurium 14028 s PB5257 PB6716 PB4836 E. coli MC1061 PB8820 PB9453 Plasmids pPROBE-NT pPB1294 (pPB-GFP) pPB1295 (pPA-GFP) pPB1296 (pGOLS-GFP) pPB1321 (pGOLS(R)-GFP) Wild-type S. enterica serov. typhimurium S. typhimurium 14028 s DgolS S. typhimurium 14028 s DgolS DcueR S. typhimurium 14028 s DgolTS-golB DgesABC hsdR2 hsdM hsdS araD139 D(ara-leu)7697 D(lac)X74 galE15 galK16 rpsL (StrR) mcrA mcrB1 E. coli MC1061 golS CmR E. coli MC1061 golTSgolB CmR repp BBR1 KmR promoterless gfp pPROBE-NT derived plasmid with the golB promoter inserted upstream the gfp gene pPROBE-NT derived plasmid with the copA promoter inserted upstream the gfp gene pPROBE-NT derived plasmid with the golS gene with its own promoter and the golB promoter inserted upstream the gfp gene pPB-GFP derived plasmid with the golS gene with its own promoter inserted upstream and in the opposite orientation regarding the PgolB::gfp construction repp15A CmR pSU8 derived plasmid with the golS promoterless sequence inserted at the multiple cloning site reppMB1 TetR lacIpMC1871 derived plasmid with the golB promoter inserted upstream the lacZ gene

Refs. or source ATCC1-14028TM (Checa et al., 2007) This work This work (Casadaban and Cohen, 1980) This work This work (Miller et al., 2000) This work This work This work This work

pSU8 pPB1322 (pGOLS-Cm) pMC1871 pPB1222 (pPgolB)

(Bartolome et al., 1991) This work Amersham (Perez Audero et al., 2010)

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(St. Louis, MO), except LB culture medium that was from DifcoTM-BD Co. (Sparks, MD). All heavy metal salts were at least of analytical grade (purity greater than 95%).

Metal Induction Assays Sensor strains were initially screened on LB-agar plates supplemented with either 40 mM AuHCl4 or 1 mM CuSO4. After overnight incubation at 378C, green uorescent colonies were visualized on a UV transilluminator [UV light induces changes in protein conformation that favors ionization of the Tyr66 residue in the chromophore resulting in green light emission by the bacteria expressing GFP (Cubitt et al., 1995)]. Metal induction assays were routinely performed in cells grown at 378C with shaking in SM9 minimal media (Checa et al., 2007) supplemented with 0.2% glucose, 0.5% casamino acids and 2 mg mL1 thiamine, except when indicated. At mid exponential phase (OD600 0.7), a 1 mL aliquot of the culture was added to test tubes containing the indicated concentration of AuHCl4 3H2O, KAu(CN)2, Auranon [3,4,5-triacetyloxy6-acetyloxymethyl oxane-2-thiolate triethylphosphanium Au(I) salt], AgNO3, CuSO4 5H2O, ZnCl2, CdCl2, HgCl2, NiSO4 6H2O, CoCl2 6H2O, FeSO4 7H2O, or sterile water. Incubation was continued for additional 3 h (unless otherwise indicated) in the same conditions, before measuring emitted uorescence or b-Galactosidase activity. After incubation, cells grown in LB were harvested by centrifugation, washed, and resuspended into an equal volume of the phosphate-buffered saline (PBS, pH 7.1) in order to minimize background uorescence exhibited by the culture media. At least ve independent experiments in triplicate were performed for each sensor bacterium and its control strain. Metal stock solutions were prepared at a high concentration (1 or 0.5 M) in distilled water and stored at 48C. Working dilutions of metals were made daily in distilled water in order to apply a 10 mL aliquot onto each test tube. Western blot analysis of GolS was performed essentially as described (Perez Audero et al., 2010), but using rabbit polyclonal anti-GolS antibodies and the ECL Plus Western Blotting Detection System (Amersham). Protein concentration was determined by Bradford assay, using bovine serum albumin as standard. Quantication by densitometry of membranes was performed using the ImageJ software (Abramoff et al., 2004).

emission wavelengths, respectively. The nal optical density at 600 nm (OD600) of each sample was also measured. The uorescence measurements ( F) were normalized following the formula:     RFU1 RFU2 F OD1 OD2 600 600 where RFU1 is the uorescence (measured in instruments arbitrary relative uorescence units) and OD1 , the nal 600 optic density determined for each sample obtained from the sensor bacteria, and RFU2 and OD2 , the same parameters 600 determined for the strain carrying the pPROBE-NT vector. Induction coefcients (IC) were calculated using the formula: IC FS/FB, where FS is the normalized uorescence value of the sensor bacteria exposed to the metal, and FB is the normalized uorescence of the biosensor cultured without metal added (background uorescence). A calibration plot for the transgenic E. coli golTSB sensor was constructed to determine the range of Au(I) concentration that gave rise to a linear relationship between the calculated IC and the amount of metal added to the cultured media. The limit of Au determination was set according to the SD of the uorescence measured for the biosensor bacteria grown in the absence of metal (Winefordner and Long, 1983). It was determined using the equation shown in the calibration plot of Fig. 4, as the concentration of Au ions (in nM or mg L1) that induced the reporter expression to a value equal to 2 ( FB 3 SD)/FB, where FB is the mean background uorescence value of the sensor and SD, the standard deviation. Microsoft Excel program was used for data management, calculations, and for performing regression analysis. b-Galactosidase assays were carried out essentially as described previously (Checa et al., 2007), except that bacterial cells were diluted 1:100 in fresh medium before applying to the testing mix. b-Galactosidase activity expressed as Miller Units (Miller, 1972) was normalized against OD600 and reaction times. IC were calculated basically as described for uorescence measurement for comparison.

Results
Construction of a Fluorescent Salmonella Au-Biosensor The Salmonella GolS sensor selectively recognizes Au(I) ions in the bacterial cytoplasm to activate transcription of genes that endow the bacteria with resistance to this heavy metal ion (Checa et al., 2007; Pontel et al., 2007). To use this system as a Au-biosensor, we constructed a reporter plasmid carrying the A. victoria gfp gene under the control of the GolS-controlled golB promoter and introduced it into S. typhimurium, the original host of the Au-responsive regulatory unit. Thus, after Au(I) is detected by the resident transcriptional regulator, the expression of the reporter gene would be induced resulting in uorescence increase. To

Measurements and Calculations For uorescence determinations, 200 mL aliquots of each sample were applied in triplicate into a 96-well at bottom black plates (Greiner Bio-One). Fluorescence was recorded using a Synergy 2 Multi-Mode Microplate Reader (BioTek) using 485 20 and 528 20 nm lters for excitation and

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analyze the sensory system, exponentially growing cells were incubated with sub-lethal concentrations of different Au compounds as well as Cu or Ag salts for 3 h, the minimal time-period to achieve maximal induction (data not shown, see also Fig. S5B). Afterwards, uorescence and nal culture OD600 were recorded. As expected, all Au compounds provoked maximal induction of the reporter gene, while Cu or Ag salts produced only minor changes in uorescence compared with the Au-driving response both in minimal and rich media (Fig. 1A and C). No statistically

Figure 2. The biosensor performance for Au detection is impaired when GolS is expressed from a multicopy plasmid. The gure shows the relative response (as IC) to increasing concentrations of KAu(CN)2 (Au) or CuSO4 (Cu) of E. coli cells or a S. typhimurium mutant strain (STM gol) harboring GolS-expression plasmids in SM9 medium. Incubation with the metals and calculations were performed as described in Fig. 1. The plasmids used in each case and a schematic representation on the relevant part of the constructions are depicted in the upper part of each gure. The normalized background values expressed as arbitrary uorescence units were as follows: 5,470 1,033 for cells carrying pGOLS-Cm and pPB-GFP; 2182 178 for those carrying pGOLS-GFP; and 2023 402 and 1373 140 for E. coli and STM gol cells harboring pGOLS(R)-GFP, respectively. The data represent the mean SD of ve independent measurements done in triplicate.

Figure 1. Selectivity of the whole-cell Salmonella uorescent biosensor to metal ions. The relative response (as IC) of either wild-type S. typhimurium cells (w-t STM) or the indicated mutant strains harboring the reporter plasmids pPB-GFP (PgolB-gfp), pPgolB (PgolB-lacZ), or pPA-GFP (PcopA-gfp) was recorded after incubation with the indicated compounds for 3 h at 378C in SM9 (A and B) or LB (C) medium (see the Materials and Methods section for details on measurements and calculations). The compounds were used at a nal concentration of 40 mM AuHCl4, 40 mM KAu(CN)2, 10 mM Auranon, 10 mM AgNO3, 35 mM CuSO4, 100 mM ZnCl2, 80 mM CdCl2, 5 mM HgCl2, 50 mM NiSO4, 120 mM CoCl2, and 25 mM FeSO4 in SM9 medium; and 80 mM AuHCl4, 80 mM KAu(CN)2, 10 mM Auranon, and 2 mM CuSO4 in LB. The concentration chosen for each compound was the highest that allowed the same growth rate as without any addition (see Fig. S1). The values for normalized background () measured as arbitrary units were as follows: 176 43 and 153 22 for PgolB-gfp on SM9 and LB, respectively; 4585 598 for PcopA-gfp; and 541 69 for PgolB-lacZ. The data represent the mean SD of ve independent measurements done in triplicate.

signicant induction was observed in the presence of other heavy metal ions (Fig. 1A) [For each metal salt, dose/ response and nal OD600 curves were done to establish the highest concentration of each metal salt required for maximal reporter induction (Fig. S1).] The accurate regulation of the reporter construction was veried in cells lacking GolS or both GolS and CueR, the homolog Cusensor that induces the expression from GolS-dependent promoters in a DgolS strain (Perez Audero et al., 2010). When the mutant cells were grown in the presence of Au ions, no metal-dependent induction was observed (Fig. 1C). Although activation by Au ions in cells grown in LB was at least threefold higher than in minimal medium (Fig. 1A and C), we choose SM9 as the culture medium for future determinations. This was primary based on the observation that there is a high uorescence background in LB and it was necessary to wash the cells twice with PBS prior to uorescence determinations, making routine determinations more laborious. The GolS-based uorescent biosensor showed a higher response to Au and Au/Cu discrimination index (dened as the ratio between the maximal induction reached by either metal) compared to a similar reporter strain that expressed the gfp gene from the CueR-controlled copA promoter from S. typhimurium (Fig. 1B). The uorescence of the GolSbased sensor increased more than 450-fold in the presence of Au ions while it exhibited a Au/Cu discrimination index of at least 100. By contrast, the CueR-based sensor bacteria showed only a 11-fold increase in uorescence by Au

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induction with a calculated Au/Cu discrimination index of <5. The uorescent Au biosensor also showed an improved response to monovalent metals compared to a similar GolSbased lacZ-reporter strain previously constructed in our laboratory (Fig. 1A). Besides being more sensitive to Au ions, the GFP reporter has the advantage of its high stability and its autouorescence, avoiding the addition of exogenous substrates to measure the response (Hakkila et al., 2002; Kohlmeier et al., 2007). Thus, performance of the biosensor is not inuenced by the kinetics of substrate uptake by the cells. In addition, the response of the Salmonella biosensor to Au was also evidenced by diffusion assays in agar plates or by uorescence microscopy (Fig. S2).

Introducing the Biosensory Device Into E. coli The above results validate the use of the GolS-based regulatory unit and provide the basis to develop Au biosensors using non-pathogenic bacterial strains. E. coli has been widely used as a host for the construction of whole-cell metal sensors (Harms, 2007; Hynninen and Virta, 2010; Magrisso et al., 2008; Robbens et al., 2010). Because the gol locus is not present in the genome of this microorganism, we initially introduced the transcriptional regulator gene in an expression plasmid. The E. coli MC1061 strain was cotransformed with the reporter pPB-GFP plasmid and the compatible medium copy number plasmid pGOLS-Cm that expresses the sensor protein from a Plac-promoter [Neither the strain nor the plasmid harbor the gene coding for the LacI repressor, thus, GolS expression was not affected by the addition of IPTG (Isopropyl-b-D-thiogalactopyranoside, data not shown)]. Although uorescence increased with Au concentration, the increments were modest compared with the Au-induction observed in the original Salmonella host (Fig. 2, see also Fig. 3C). Furthermore, there was a signicant induction of the reporter with the addition of Cu, which resulted in a low Au/Cu discrimination index. We also cloned golS under the control of its own promoter in the pPB-GFP plasmid, either preserving the original arrangement of the gol locus in S. typhimurium, or in the orientation opposite to the PgolB::gfp construction (Fig. 2). These constructions did not improve the biosensor performance. We hypothesized that the differences in metal response between the original Salmonella reporter strain and the E. coli cells carrying GolS-expression plasmids are caused by different availability of the sensor protein; i.e., GolS is expressed in Salmonella from its chromosomal single-copy gene and its concentration under non-inducing conditions is very low (Perez Audero et al., 2010), while in E. coli there are several copies of the golS gene that would drive its expression to a signicant level even in the absence of metal ions. Indeed, we observed the same pattern of induction of GFP expression than in E. coli when a S. typhimurium strain deleted in the gol locus (to mimic the E. coli conditions) was transformed with the pGOLS(R)-GFP plasmid (Fig. 2).

Figure 3. Construction and evaluation of golS-chromosomally encoded transgenic E. coli strains. (A) Genetic organization of the site chosen for the insertion of the golS-cat or golTS-golB-cat DNA fragments in the E. coli chromosome (EC). The same region in the S. typhimurium LT2 genome (STM) is also shown. (B) GolS immunodetection in whole-cells extracts obtained from wild-type S. typhimurium, or the transgenic E. coli golTSB or golS strains grown in LB with or without the addition of 0.1 or 1 mM KAu(CN)2. Whole cell extracts were analyzed by SDS-PAGE, transferred to nitrocellulose. GolS was detected by rabbit polyclonal anti-GolS antibodies. The arrows on the right indicate the immunoreactive band corresponding to either wild-type GolS or a truncated form of the sensor observed in the E. coli extracts. (C) Response of the Salmonella or the E. coli biosensors to increasing concentrations of KAu(CN)2 (Au) or CuSO4 (Cu). Incubation with the metals in SM9 medium and calculations were performed as described in Fig. 1. The normalized background values expressed as arbitrary uorescence units were 176 43, 352 18, and 270 70 for STM, EC golTSB, and EC golS biosensors, respectively. The data represent the mean / standard deviation of ve independent measurements done in triplicate. Single Copy Chromosomal Expression of golS Improves Biosensor Performance for Au Detection To emulate the conditions in which the Au-responsive regulatory unit operates in the original host, we introduced in the chromosome of the E. coli MC1061 strain, PCRamplied Salmonella DNA fragments harboring either the golS gene under the control of its own promoter or the GolScontrolled golTS-golB cluster. The DNA fragments were

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Doseresponse curve of the transgenic E. coli golTSB biosensor to Au(I). Response (as IC) versus KAu(CN)2 concentration. Incubation and calculations were performed as described in Fig. 1. The data represent the mean SD of at least ten independent measurements done in triplicate. The tted dose/response curve for the data that shows a linear relationship is showed in the insert. The lineal regression equation and the value of the coefcient of determination (R 2) for the calibration plot (line) obtained using the Excel program (Microsoft) are also indicated. The normalized background values were 352 18 arbitrary uorescence units.

Figure 4.

cells (Fig. 3B and C). At high Au concentrations, maximal activation in the transgenic biosensors was slightly lower than in the original host (Fig. 3C), probably reecting differences between the bacterial strains in envelope permeability or metal handling that affect intracellular Au(I) availability. Among the transgenic E. coli biosensors, the golTSB reporter strain showed both a copper response and a calculated Au/Cu discrimination index closer to the values obtained in Salmonella (Fig. 3C); i.e., the Au/Cu discrimination index calculated at a concentration 10 mM of either metal ion was approximately 120 for the E. coli biosensor and 200 for the Salmonella biosensor (dose response curves for the E. coli and Salmonella Au-biosensors are shown in Fig. S3.). These results indicate that expression of the innate GolS-regulated genes golT and golB, coding for a P-type ATPase and a small metal-binding protein, respectively, together with the Au-sensor facilitate metal ion detoxication and improve biosensor performance for Au detection in the E. coli cells. The response of the transgenic E. coli biosensor to other metal salts was also tested in order to verify a similar behavior of the reporter construction in the new host (Fig. S4).

Calibration of the Transgenic E. coli Au-Biosensor Growth phase and cells nutritional condition may inuence not only the reporter expression, but also the envelope status, affecting ion permeability (Charrier et al., 2011; Kohlmeier et al., 2007). Thus, we evaluated the response of the transgenic E. coli golTSB reporter strain to Au and Cu ions under different conditions. Cells were grown until midexponential phase in minimal SM9 medium supplemented with different carbon sources or in LB. As was previously observed for the Salmonella biosensor, maximal induction by Au was attained in rich medium (Fig. S5A). For cells grown in minimal medium, we observed an improved Auresponse with similar Au/Cu discrimination index than in LB by using glucose-supplemented SM9. Like in Salmonella, we selected this medium for calibration of the bacterial sensor. In order to optimize metal exposure time, the transgenic biosensor was exposed to Au ions for different time periods. Maximal GFP expression was obtained after 3 h incubation with the metal regardless the Au concentrations used (Fig. S5B). Longer incubation times did not signicantly increase the response; although uorescence was stable for at least 24 h. We performed calibration dose/response curves of the transgenic E. coli golTSB biosensor in a wide range of Au concentrations. Fluorescence increased with Au(I) concentration up to 470 nM (Fig. 4). No further induction was observed at higher concentrations. At a concentration of >80 mM Au, uorescence decreased sharply because of metal toxicity (Fig. 4, see also Fig. S3B). The limits for Au detection, i.e., the lowest gold concentration that produce a detectable increase in uorescence compared to the background (in this case a 2.31-fold induction in uorescence,

inserted in the ampH-sbmA intergenic region (at position 395486 in the chromosome of the E. coli W3110 strain; see Fig. 3A). This region was selected because the array of genes is similar in both S. typhimurium and E. coli, and it is located close to the thrW tRNA gene, the site of insertion of the $45 kb Salmonella specic region harboring the gol locus (http://www.sanger.ac.uk/Projects/Salmonella; Checa and Soncini, 2011). First, we veried the GolS autoregulation in both the golS and the golTS-golB (or golTSB) transgenic E. coli strains, as occurs in Salmonella. By western blot analysis, we observed that GolS levels increased with Au concentration in both strains, while there was no detectable expression of the sensor protein under non-inducing conditions (Fig. 3B). At low Au concentrations the cytoplasmic accumulation of GolS in the transgenic E. coli strains was 35-times lower than in Salmonella. Interestingly, at 1 mM KAu(CN)2, we observed that the levels of GolS increased up to threefold in the E. coli strains compared to wild-type Salmonella (Fig. 3B). However, the sensor protein appeared as a double immunogenic band in the E. coli extracts, probably evidencing proteolytic processing of the heterologous protein in this bacterial host. The golS and golTSB transgenic E. coli cells were transformed with the pPB-GFP reporter plasmid to evaluate their response to increasing concentrations of Au or Cu. The pattern of Au-directed induction of GFP expression in both transgenic strains was similar to the response observed in Salmonella and correlates well with the level of GolS in the

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n 8, coefcient of variation 5.23%) was 33.23 nM Au(I), equivalent to 6.54 mg L1 (or ppb) (This was calculated by using the equation of the linear calibration curve shown in Fig. 4 and the equation provided in the Materials and Methods section). These results indicate that the transgenic E. coli golTSB strain is appropriate to be used as a Au detector at least under laboratory conditions.

Discussion
The unique property of the Salmonella GolS protein to favor the binding of Au over Cu or Ag ions allowed us to design and construct highly sensitive and selective uorescent bacterial biosensors that serve as robust Au detectors. We used both the gol-resident S. typhimurium strain and a transgenic E. coli strain expressing the Salmonella gol locus as hosts for the reporter construction (Fig. 3). The GolS-based bacterial sensors selectively recognize Au ions and have sensitivity for detecting the toxic metal at least one order of magnitude higher than a similar uorescent biosensor based on the CueR/PcopA regulatory unit (Fig. 1). At standard assay conditions in the laboratory, the response of the transgenic E. coli biosensor to Au ions increases with metal concentration (up to 470 nM), with a threshold for detection estimated in $33 nM or 6.5 mg L1 (Fig. 4). These values are well below the concentrations typically found nearby gold-rich deposits (Butt and Hough, 2009; Southam et al., 2009), which a priori make these bacterial detectors appropriate for this application. The high sensitivity of the built Au-biosensors depends on the natural afnity of GolS to bind the toxic ion (Checa et al., 2007). Although not determined for GolS, the binding afnity of Cu(I), Ag(I), or Au(I) determined for the related copper sensor, CueR, has been estimated in the zeptomolar (1021 M) range (Changela et al., 2003). A number of in vivo and in vitro experiments performed with the Salmonella GolS and its xenolog from Cupriavidus metallidurans, CupR, indicate that Au selectivity in these sensors is ensured by lowering their afnity for Cu(I) in comparison to the Cusensors, while maintaining or even increasing the afnity for Au(I) (Checa et al., 2007; Jian et al., 2009). The improved response of the sensor/regulatory unit to Au ions offsets the assumed small sensitivity of GFP used here as the reporter. The election of the uorescent reporter protein instead of its luminescent counterparts is based mainly on the possibility to perform single-cell analysis in the laboratory or to couple the sensor bacteria to portable instruments for eldwork detection (Hakkila et al., 2002; Kohlmeier et al., 2007; Tombolini and Jansson, 1998). Besides, gfp-based reporters are less sensitive to growth conditions, nutritional status or the presence of inhibitors in samples, and they do not require the addition of any substrate to develop a detectable signal, in contrast to other reporter devices. In particular, the gfp cassette used here encodes a mutant GFP form with enhanced solubility and uorescence, and a red shift in the excitation spectrum that improves determinations (Miller et al., 2000).

The most challenging eld for applications of the designed biosensor is the evaluation of Au placers as an inexpensive, yet effective alternative to analytical tests (Harms, 2007; Magrisso et al., 2008). The concentration of the metal in auriferous soils usually exceeds 100 mg L1 (or ppb), and it is combined with silver and copper among other metals (Hough et al., 2009). The performance under laboratory conditions of the bacterial Au detector designed here is promising in terms of sensitivity and selectivity (Figs. 3 and 4). Thus, based on the results shown in Figures S3 and S4, the biosensors will be able to detected Au at concentrations closed to the calculated detection limit even in the presence of sublethal concentration of Cu or Ag. Even though the biosensor detects only the biologically available fraction of Au, which is usually low in soil samples, we predict that it would be useful in bio-mining (Reith et al., 2010; Valenzuela et al., 2006). The conditions found in deposits and lixiviates are usually unfavorable for microbial survival. Therefore, we expect that it would be possible to transfer this sensor/response construction to more resistant bacterial species (Reith et al., 2010). One important observation made in this work is that, in contrast to a previously designed metal sensor that express the sensor/response unit from a plasmid (Harms, 2007; Magrisso et al., 2008; and references there in), proper performance for the Au biosensors requires the sensor encoding gene in a single copy in the chromosome (Figs. 2 and 3), as it occurs in the original Salmonella host. We are currently evaluating the suitability to transfer the reporter construction to C. metallidurans CH43, a b-proteobacterium found forming biolms on Au grains (Reith et al., 2010). This bacterium harbors a Au-inducible gol-like cup cluster in its chromosome that codes for the P1B-type ATPase CupA, the metal chaperone CupC and the GolSxenolog CupR that directs the response to Au, regulating transcription from gol-like promoters (Jian et al., 2009; Perez Audero et al., 2010). In sum, the Au-selective bacterial biosensors presented here, are the rst step for the development of rapid, specic, and inexpensive screening tools to detect the precious metal in gold deposits.
We thank E. Garca Vescovi, M. E. Castelli, L. B. Pontel, and M. M. Ibanez for critical reading and comments on the manuscript, S. E. Lindow for its kind gift of plasmid pPROBE-NT, and J. Pellegrino for helping us at the initial steps of uorescence determinations. This work was supported by grants from Agencia Nacional de Promocion Cientca y Tecnologica and from the National Research Council (CONICET) to F. C. S. and S. K. C. S. C. is a fellow of the CONICET, and F. C. S. and S. K. C. are career investigators of the same institution. F. C. S. is also a career investigator of the Rosario National University Research Council (CIUNR).

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