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Whole-cell bacterial sensors for the monitoring of phosphate bioavailability
Journal of Microbiological Methods 55 (2003) 221 – 229 www.elsevier.com/locate/jmicmeth
Whole-cell bacterial sensors for the monitoring of phosphate bioavailability Marie-Andre´e Dollard, Patrick Billard * U.R. Ecotoxicite´, Biodiversite´, Sante´ Environnementale, Universite´ de Metz, Campus Bridoux—Rue du Ge´ne´ral Delestraint, 57070 Metz, France Received 27 November 2002; received in revised form 14 April 2003; accepted 15 April 2003
Abstract A phosphate sensor plasmid was constructed, in which the inducible promoter of the alkaline phosphatase gene (phoA) from Escherichia coli is fused to the bioluminescence genes from Vibrio fischeri. The reporter construct was introduced into E. coli MG1655 and the rhizosphere coloniser Pseudomonas fluorescens DF57, which produced light in a dose-dependent manner when exogenous phosphate concentrations fell below 60 and 40 AM, respectively. These strains also responded to various organic and inorganic phosphorus compounds. Their ability to distinguish the bioavailable portion of phosphate in standard solution was demonstrated using different phosphate ligands. When applying the bioassay to wastewater samples, luminescence patterns correlated with phosphate concentrations determined by standard chemical procedure. These results indicated that phoA::luxbased bacterial sensors may serve as tools for the assessment of phosphate bioavailability. D 2003 Elsevier B.V. All rights reserved. Keywords: Bacterial sensors; Bioavailability; Bioluminescence; phoA; Phosphate; Reporter gene bioassay
1. Introduction Increased phosphate concentration of water bodies has long been recognised as one of the major cause of eutrophication of lakes and rivers. Pollution by inorganic phosphate is mainly due to the wide use of fertilizers and phosphated detergents and may lead to many water quality problems such as those related to algal bloom formation (Yeoman et al., 1988). Thus, there is a need for simple and reliable methods for
* Corresponding author. Tel.: +33-387-37-85-13; fax: +33-38737-85-12. E-mail address: [email protected] (P. Billard).
routine phosphate analysis of water and soil samples. Phosphate measurements are also required in other areas such as clinical diagnosis, where its concentration in body fluids can be used to monitor several diseases (Kawasaki et al., 1989; Male and Luong, 1991) and food analysis because of the deleterious effects of phosphate-rich diets on human health (Watanabe et al., 1988). Besides the classical colorimetric methods (Fiske and Subbarow, 1925; Murphy and Riley, 1962), which are complex and time consuming, a number of alternative strategies of phosphate (and/or phosphorus) determination based on ion-selective electrodes or enzyme sensors (for a review, see Engblom, 1998) or capillarity electrophoresis (PantsarKallio and Manninen, 1995) have been developed in
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the last decades. Complementary to these approaches, sensitive bioassays relying on algal or bacterial growth (Ekholm and Krogerus, 1998; Lehtola et al., 1999) have been described that allow the evaluation of phosphorus bioavailability in water samples. Recent advances in the application of reporter gene technology to environmental monitoring purposes led to the development of rapid bioassays to assess the bioavailability of specific pollutants in complex environments. Most of them use luminescent bacterial sensors, in which the promoterless lux genes from Vibrio harveyi or Vibrio fischeri are inserted downstream a pollutant-responsive gene, so that light is produced when the recombinant bacteria is exposed to the cognate compound. A number of strains have been designed to detect organic and metal contaminants (Billard and DuBow, 1998; Ko¨hler et al., 2000; Daunert et al., 2000; Keane et al., 2002) and also to assay the availability of several nutrients including nitrate (Prest et al., 1997), iron (Durham et al., 2002), and phosphorus (Schreiter et al., 2001; Gillor et al., 2002). In the latter case, the inducible alkaline phosphatase gene (phoA) from a cyanobacterium strain was used as the phosphorus-sensing element. The resulting sensor strain detected phosphate with high sensitivity and was proposed as a new tool for investigating the relationships between phosphorus bioavailability and the fate of cyanobacterial population in freshwater environments. The phoA gene belongs to the Pho regulon, a system found in many taxonomic groups of microorganisms, that mediates the cell response to deficiency of exogenous phosphate. In Escherichia coli, the expression of the Pho regulon is triggered by phosphate-limiting conditions, leading to the induction of phoA and a set of other genes whose products allow a better phosphate uptake or phosphorus supply from other environmental sources (Wanner, 1996). In a preliminary study, we tested the feasibility of detecting phosphate using the reporter gene technology (Riether et al., 2000). The phoA promoter from E. coli was fused to the promoterless luxCDABE operon in the pSA-derived vector pUCD615 (Rogowsky et al., 1987). This plasmid replicates in a variety of Gram-negative bacteria and has proven to be useful in bacterial constructs for the monitoring of environmental pollutants (Selifonova et al., 1993; Vollmer et al., 1997; Riether et al., 2001). An E. coli strain
carrying this plasmid-based phoA::luxCDABE transcriptional fusion was found to emit light in a dosedependent manner upon phosphate starvation. In this communication, we report on the construction and characterization of the above phosphate sensor system. In addition to E. coli, we used the more environmentally relevant Pseudomonas fluorescens DF57 strain (Kragelund et al., 1995) as a host for the reporter gene construct. The resultant sensor strains were tested for their ability to report biologically accessible concentrations of phosphate in both standard solutions and wastewater samples using a simple and rapid bioassay.
2. Materials and methods 2.1. Bacterial strains and culture conditions E. coli DH10B (Life Technologies) was used for cloning experiments and propagation of plasmids. This strain was also the host for plasmid pUCD607, which contains the luxCDABE genes from V. fischeri under the control of the tetracycline resistance promoter (Shaw and Kado, 1986) and confers a constitutive bioluminescence phenotype. E. coli MG1655 (wild type) and P. fluorescens DF57 (Kragelund et al., 1995) were the recipients for the reporter plasmid pPHO-lux, the resulting phosphate sensor strains were used in bioluminescence assays. E. coli strains were cultured aerobically at 37 jC in Luria Broth (LB) or in MOPS minimal medium (Neidhardt et al., 1974). P. fluorescens was grown aerobically at 28 jC in either LB or Davis Minimal Medium (DMM) (Neidhardt et al., 1974). When appropriate, media were supplemented with ampicillin (50 Ag/ml), kanamycin (20 Ag/ml), or chloramphenicol (20 Ag/ml). 2.2. Construction of plasmid pPHO-lux General procedures for strain and plasmid construction were performed according to Sambrook et al. (1989). The plasmid pPHO-lux containing a transcriptional fusion of the phoA gene to the luxCDABE operon was constructed in a manner similar to that previously described (Riether et al., 2001). The phoA promoter region of E. coli was amplified from strain MG1655 genomic DNA by PCR using modified
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upstream primer pPHO1 (5V-CCGGATCCTGACCGACGATACGGAG-3V) that created a BamH1 site (in italics sequence) at position 244 with respect to the phoA start codon and unmodified downstream primer pPHO2 (5V-GTCACCGAATTCAGTGAGGC3V) containing an EcoRI site at position + 720. Amplification was carried out with a PTC-100 thermocycler (MJ Research), with an initial denaturation step (94 jC for 5 min) followed by 30 cycles of 94 jC for 30 s, 55 jC for 45 s, and 72 jC for 1 min. A final extension was performed for 5 min at 72 jC. The resulting PCR product was digested with BamH1 and EcoRI and ligated into BamH1 – EcoRI-digested pUCD615 (Rogowsky et al., 1987), to yield plasmid pPHO-lux, in which the complete luxCDABE operon from V. fischeri was controlled by the phoA promoter. Plasmid pPHO-lux was introduced into E. coli MG1655 and P. fluorescens DF57 by electroporation (Dower et al., 1988; Itoh et al., 1994).
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2.3. Preparation of cells and luminescence measurements Overnight MOPS cultures of E. coli MG1655/ pPHO-lux were diluted to an OD600 of 0.1 in fresh MOPS and grown with shaking until the exponentially growing phase was reached (OD600 of 0.4 –0.6). The cells were harvested by centrifugation at 6000 g for 10 min at room temperature, washed twice with one volume of MOPS minimal medium without phosphate, and then suspended in the same medium to obtain a final OD600 of 10. The same protocol was applied to DMM cultures of P. fluorescens DF57/ pPHO-lux. The positive control E. coli DH10B/pUCD607 strain was prepared as follows: an overnight LB culture of the bacteria was harvested by centrifugation at OD600 of 3– 4, washed with one volume of 10-fold concentrated GGM medium (Riether et al., 2001). The
Fig. 1. Kinetics of phoA::luxCDABE induction as a function of phosphate concentration in E. coli/pPHO-lux (A) and P. fluorescens/pPHO-lux (B). Values are means F S.D. (n = 3).
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bacteria were then suspended in the same medium to an OD600 of 2 before the measurements. In the standard induction assay, 10 Al of freshly prepared cell suspension were added to 90 Al of test solution in opaque white 96-well microtiter plates (Dynatech, Germany). The plates were immediately placed in a temperature-controlled (26 jC) microtiter plate luminometer (LUMIstar, BMG LabTechnologies, Germany), and bioluminescence was recorded in intervals of 20 min for at least 3 h, each sample being measured for 10 s. Before each measurement cycle, the plates were shaken for 1 min in the luminometer. Raw luminescence values were presented in the instrument’s arbitrary relative light units (RLU). In some cases, we also calculated the induction coefficient, which is equal to the luminescence in a samplecontaining well, divided by that in well containing uninduced cells (that is, in the presence of 0.2 mM K2HPO4) at the same time point. The minimum detection threshold was determined as the concentration at which the induction coefficient was 2. For phosphate bioavailability experiments, results were expressed as the percentage of the luminescence measured in a control sample containing no phosphate or ligand. All experiments were conducted in triplicate and repeated at least two times. Luminescence in triplicates varied by less than 15%, whereas variations between experiments normally did not exceed 40%.
ison, total phosphorus and orthophosphate concentrations were determined in filtered samples according to standard methods (AFNOR NF T 90-023, 1983).
3. Results and discussion 3.1. Kinetics of phoA::luxCDABE induction in E. coli and P. fluorescens Representative luminescence patterns of E. coli/ pPHO-lux and P. fluorescens/pPHO-lux when incubated with variable amounts of orthophosphate are shown in Fig. 1. E. coli responded to phosphate deprivation by a dose-dependent increase in light emission measurable after only 40 min of incubation. However, because of the transient increase in background luminescence in the first part of the assay, extended incubation times gave higher induction coefficients. As a compromise between sensitivity
2.4. Wastewater samples Water samples (raw sewage and final effluent) were collected from a wastewater treatment plant in Metz, France. Suspended solids were removed by centrifugation (6000 g for 10 min), and the resulting supernatants were filtered through a 0.45-Am filter. These samples were diluted with distilled water to final concentrations of 5 – 75% of the original and then assayed for bioavailable phosphate according to the above standard induction assay. A control assay using E. coli DH10B/pUCD607 was conducted in parallel to assess sample toxicity and matrix effect. Results were presented as the percentage of luminescence relative to distilled water controls. Apparent bioavailable phosphate concentrations in water samples were calculated by comparing luminescence values in diluted samples with that of a standard calibration curve. For compar-
Fig. 2. Response of E. coli/pPHO-lux (A) and P. fluorescens/pPHOlux (B) to different phosphorus compounds. K2HPO4 ( y); tripolyphosphate (E); h-glycerophosphate (5); glucose-6-phosphate (o); pyrophosphate ( ). Values presented were measured after 140 and 120 min incubation for E. coli and P. fluorescens, respectively. Error bars represent the S.D. for n = 3.
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and response time, luminescence values measured after 140 min of induction were chosen for further experiments. At this time point, the detection threshold, defined as the minimal phosphate concentration causing at least a twofold increase over background luminescence, was 60 AM (see Fig. 2A). In P. fluorescens, the expression of the phoA::luxCDABE fusion was also dependent on external phosphate concentration, with maximal induction occurring after 120 min incubation. The phosphate detection threshold at this incubation time (40 AM; see Fig. 2B) was slightly lower than that estimated for E. coli. On the other hand, the induction pattern differed from that of E. coli by a lag before induction, lower luminescence intensity, and a stable baseline throughout the assay. The heterologous expression of the E. coli phoA promoter in P. fluorescens is not so surprising and is in agreement with previous observations suggesting similarities in the control of the PHO regulon between the two bacteria (Kragelund et al., 1995). Similar phosphate-dependent light emission was also ob-
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served when Acinetobacter sp. strain ADP1 was used as a host for pPHO-lux (not shown). 3.2. Analyte spectrum In addition to phosphate, we examined the response of our reporter strains when incubated with two inorganic phosphates (pyrophosphate and tripolyphosphate) and two organic phosphate esters (h-glycerophosphate and glucose-6-phosphate), which potentially serve as environmental phosphorus source for microorganisms. As shown in Fig. 2, all four compounds were able to inhibit light emission by the two reporter strains in a concentration-dependent manner. This indicated that they served to supply cells with phosphate, probably via breakdown by periplasmic phosphatases, and thus repressed the expression of the phoA::luxCDABE fusion. For E. coli, the detection thresholds of the tested compounds appeared to decrease in the following order: h-glycerophosphate>pyrophosphate>tripolyphosphate>glucose-6-phosphate. Even the latter
Fig. 3. Influence of different phosphate ligands on the response of E. coli/pPHO-lux (A, C) and P. fluorescens/pPHO-lux (B, D). Panels (A) and (B): inhibition of luminescence of the sensors strains by ligands in a phosphate-free medium. Panels (C) and (D): effect of increasing concentrations of ligands on the response of the sensor strains in an assay medium containing 100 AM K2HPO4. FeCl3 ( ); Al2(SO4)3 (n); LaCl3 (o). Luminescence values 140 and 120 min after induction were used for E. coli and P. fluorescens, respectively. Error bars represent the S.D. for n = 3.
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compound, however, repressed the phoA expression at concentrations 20-fold higher than that required with phosphate, indicating that this sensor strain primary reports the bioavailability of phosphate. Some substrates such as pyrophosphate and tripolyphosphate seemed to produce a biphasic response curve, with intermediate plateau values followed by a further reduction in the luminescence level. Since no specific transporter system has been reported for these compounds in E. coli (Wanner, 1996), they may enter the cell either through a nonspecific pathway or after hydrolyse by periplasmic phosphatase(s). Intermediate luminescence plateau values may therefore reflect the saturation of one transport/breakdown system followed by the setup of a second nonspecific one at higher substrate concentrations. When compared to E. coli, the response of P. fluorescens was characterized by smaller dynamic ranges for phoA induction, the absence of intermediate plateau for pyrophosphate and tripolyphosphate, and a strikingly high detection threshold for glucose-6-phosphate (20 mM). The latter findings illustrate the contrasting ecophysiology of the two reporter strains and probably reflect differences in their phosphatase activity profiles.
concentration of 100 AM K2HPO4, which is high enough to ensure a basal luminescence emission. Results were expressed as the percentage of the luminescence measured in a control sample containing no phosphate or ligand. Response values near 1 therefore indicated the absence of (bioavailable) phosphate. As expected, addition of these compounds reduced the bioavailability of phosphate to the two biosensor strains, as judged by the progressive increase in the luminescence signal (Fig. 3C and D). LaCl3 appeared to be the most effective phosphate ligand, a concentration of 75 AM LaCl3 (LaCl3/Pi ratios = 0.75) being sufficient to make phosphate totally unavailable to E. coli. For higher LaCl3/Pi ratios, however, the luminescence level decreased. Considering the results presented in Fig. 3A and B, this effect can probably be attributed to the toxicity of lanthanum ions. Similar patterns were observed for FeCl3 and Al2 (SO4)3, the induction level and subsequent lumines-
3.3. Phosphate bioavailability One key advantage of whole-cell biosensors over conventional methods for determination of environmental contaminants concentrations is their ability to assess the bioavailable or bio-active fraction of these compounds. To determine whether our sensor strains were able to distinguish available and immobilised forms of phosphate, bioassays were conducted in the presence of three known phosphate ligands: ferric and aluminium salts [FeCl3, Al2(SO4)3] that are commonly used in wastewater treatment to precipitate phosphorus and lanthanum (LaCl3), whose potency as alternative phosphate binder for the management of hyperphosphataemia has been evaluated (Zhu et al., 2002). To assess the potential toxicity of the ligands, the two reporter strains were exposed to increasing concentrations of these compounds in a phosphatefree medium (Fig. 3A, B). In both cases, lanthanum salts proved to be the most toxic ligand, followed by aluminium and iron salts. For bioavailibility experiment, varying concentrations of the phosphate ligands were added to an assay medium containing a fixed
Fig. 4. Luminescence response displayed by E. coli/pPHO-lux (o), P. fluorescens/pPHO-lux (E), and the control strain E. coli/ pUCD607 (n), to dilutions of wastewater samples from a treatment plant. Panel (A): influent. Panel (B): effluent. Values presented were measured after 140 and 120 min incubation for E. coli and P. fluorescens, respectively. Error bars represent the S.D. for n = 3.
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cence decrease being a balance between the effectiveness of phosphate complexation and toxicity of the ligands to each strain.
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was effectively removed in the treatment facility to reach concentrations far below the detection thresholds of the bioassay, as also confirmed by data obtained from chemical analysis (Table 1).
3.4. Application of the bioassay to analyse wastewater samples 4. Conclusions To test the usefulness of our reporter strains in more environmentally relevant samples, bioassays were performed on water samples collected from a wastewater treatment plant (Metz, France). Fig. 4 shows the bioluminescence of the two strains exposed to different dilutions of filtered raw sewage (influent) and treated effluent samples. A decrease in light production was observed with the two reporter strains when increasing influent concentrations (Fig. 4A). This suggested that influent sample contained sufficient phosphate to repress, at least in part, phoA expression in the reporter strains. Alternatively, the same decrease in light production could be the result of increase toxicity. To test this possibility, a control assay using E. coli DH10B/ pUCD607 was conducted in parallel. No significant reduction in luminescence was detected whatever the tested dilutions, ruling out toxicity and matrix effects associated with the influent sample. We therefore calculated the apparent bioavailable phosphate concentrations in influent for both E. coli/pPHO-lux and P. fluorescens/pPHO-lux, by comparing luminescence values in diluted sample with that of calibration curves. Results presented in Table 1 suggested that phosphate content of the raw sewage, as measured by chemical method, is only partially available to E. coli. By contrast, the phosphate level detected by P. fluorescens corresponded to that defined chemically. No dilution-dependent induction was observed with the effluent sample (Fig. 4B), indicating that phosphate Table 1 Phosphorus concentration in wastewater samples measured by the phosphate reporter strains or by standard chemical method Bioavailable phosphate (AM)
Chemically determined phosphorus (AM)
E. coli/ P. fluorescens/ Orthophosphate Total pPHO-lux pPHO-lux phosphorus Influent 110 F 30a 160 F 40a Effluent < 10b < 10b a b
161 0.2
Data represent the average F S.D. (n = 3). Lowest detection limit.
178 0.7
The above findings are consistent with those previously reported by Schreiter et al. (2001), whose CyanoSensor (a Synechococcus reporter strain carrying a chromosomal phoA::luxAB fusion) was shown to reflect the bioavailability of different phosphorus sources. The sensitivity of phosphate detection with our sensor strains is far lower than that reported for the CyanoSensor, thus precluding their use for the monitoring of environments with low phosphorus content, such as oligotrophic lakes or drinking water. However the plasmid-based reporter system used here provides the advantage to give information on phosphate concentration in a relatively short time, without the need for illumination as required for cyanobacterial cultures. In addition, since the phoA regulation mechanism appears to be functionally conserved in many bacteria, one might expect that introduction of the pPHO-lux plasmid in bacterial hosts with contrasting ecophysiology will generate new phosphate reporter strains. Depending upon their performance, one or more such strains can therefore be chosen for a specific application. For example, P. fluorescens appears better suited than E. coli for the semiquantitative measurement of phosphate in wastewaters and to control the efficiency of its removal in treatment plants. Even if chemical methods to determine phosphate are accurate and sensitive, they do not provide clear information on the availability of this element to organisms. The same is true for organic and inorganic phosphorus-containing compounds, whose bio-accessibility cannot be monitored in a real-time scale via chemical approaches. Our results from phosphate bioavailability experiment showed that the two reporter strains did not detect the portion of phosphate that is complexed with the ligands. Similarly, the accessibility of different phosphorus sources could be evaluated and, for some of them, appeared to vary between the two bacteria. In general, the data presented here clearly indicate the applicability of the phoA::lux concept to monitor phosphorus bioavailability. The assay is sim-
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ple, rapid and may be exploited for different purposes such as to assess the strength of new phosphatebinding compounds or to study conditions that affect phosphate and/or phosphorus-containing compounds’ bioavailability in environmental samples, in combination with positive control biosensors to rule out toxicity problems. Nevertheless, for such practical applications, the approach still requires some improvements. For example, plasmid instability and possible variations in plasmid copy number in the cells are factors that may alter the assay performance. One possibility to overcome this problem and provide a more stable system would be the integration of the phoA::lux fusion into the host strains chromosome. Future investigations will also address the lyophilisation or immobilisation of the reporter strains in polymeric matrix, which will limit luminescence variations between experiments and allow the design of an easyto-use test for bioavailable phosphate.
Acknowledgements We thank N. Babic for excellent technical assistance and P. Poupin for comments on this manuscript. The generous gifts of P. fluorescens strain DF57 by O. Nybroe, and plasmids pUCD615 and pUCD607 by C.I. Kado are acknowledged. Part of this work was supported by NANCIE and CIRSEE-Lyonnaise des Eaux.
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Whole-cell bacterial sensors for the monitoring of phosphate bioavailability Marie-Andre´e Dollard, Patrick Billard * U.R. Ecotoxicite´, Biodiversite´, Sante´ Environnementale, Universite´ de Metz, Campus Bridoux—Rue du Ge´ne´ral Delestraint, 57070 Metz, France Received 27 November 2002; received in revised form 14 April 2003; accepted 15 April 2003
Abstract A phosphate sensor plasmid was constructed, in which the inducible promoter of the alkaline phosphatase gene (phoA) from Escherichia coli is fused to the bioluminescence genes from Vibrio fischeri. The reporter construct was introduced into E. coli MG1655 and the rhizosphere coloniser Pseudomonas fluorescens DF57, which produced light in a dose-dependent manner when exogenous phosphate concentrations fell below 60 and 40 AM, respectively. These strains also responded to various organic and inorganic phosphorus compounds. Their ability to distinguish the bioavailable portion of phosphate in standard solution was demonstrated using different phosphate ligands. When applying the bioassay to wastewater samples, luminescence patterns correlated with phosphate concentrations determined by standard chemical procedure. These results indicated that phoA::luxbased bacterial sensors may serve as tools for the assessment of phosphate bioavailability. D 2003 Elsevier B.V. All rights reserved. Keywords: Bacterial sensors; Bioavailability; Bioluminescence; phoA; Phosphate; Reporter gene bioassay
1. Introduction Increased phosphate concentration of water bodies has long been recognised as one of the major cause of eutrophication of lakes and rivers. Pollution by inorganic phosphate is mainly due to the wide use of fertilizers and phosphated detergents and may lead to many water quality problems such as those related to algal bloom formation (Yeoman et al., 1988). Thus, there is a need for simple and reliable methods for
* Corresponding author. Tel.: +33-387-37-85-13; fax: +33-38737-85-12. E-mail address: [email protected] (P. Billard).
routine phosphate analysis of water and soil samples. Phosphate measurements are also required in other areas such as clinical diagnosis, where its concentration in body fluids can be used to monitor several diseases (Kawasaki et al., 1989; Male and Luong, 1991) and food analysis because of the deleterious effects of phosphate-rich diets on human health (Watanabe et al., 1988). Besides the classical colorimetric methods (Fiske and Subbarow, 1925; Murphy and Riley, 1962), which are complex and time consuming, a number of alternative strategies of phosphate (and/or phosphorus) determination based on ion-selective electrodes or enzyme sensors (for a review, see Engblom, 1998) or capillarity electrophoresis (PantsarKallio and Manninen, 1995) have been developed in
0167-7012/03/$ - see front matter D 2003 Elsevier B.V. All rights reserved. doi:10.1016/S0167-7012(03)00164-7
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the last decades. Complementary to these approaches, sensitive bioassays relying on algal or bacterial growth (Ekholm and Krogerus, 1998; Lehtola et al., 1999) have been described that allow the evaluation of phosphorus bioavailability in water samples. Recent advances in the application of reporter gene technology to environmental monitoring purposes led to the development of rapid bioassays to assess the bioavailability of specific pollutants in complex environments. Most of them use luminescent bacterial sensors, in which the promoterless lux genes from Vibrio harveyi or Vibrio fischeri are inserted downstream a pollutant-responsive gene, so that light is produced when the recombinant bacteria is exposed to the cognate compound. A number of strains have been designed to detect organic and metal contaminants (Billard and DuBow, 1998; Ko¨hler et al., 2000; Daunert et al., 2000; Keane et al., 2002) and also to assay the availability of several nutrients including nitrate (Prest et al., 1997), iron (Durham et al., 2002), and phosphorus (Schreiter et al., 2001; Gillor et al., 2002). In the latter case, the inducible alkaline phosphatase gene (phoA) from a cyanobacterium strain was used as the phosphorus-sensing element. The resulting sensor strain detected phosphate with high sensitivity and was proposed as a new tool for investigating the relationships between phosphorus bioavailability and the fate of cyanobacterial population in freshwater environments. The phoA gene belongs to the Pho regulon, a system found in many taxonomic groups of microorganisms, that mediates the cell response to deficiency of exogenous phosphate. In Escherichia coli, the expression of the Pho regulon is triggered by phosphate-limiting conditions, leading to the induction of phoA and a set of other genes whose products allow a better phosphate uptake or phosphorus supply from other environmental sources (Wanner, 1996). In a preliminary study, we tested the feasibility of detecting phosphate using the reporter gene technology (Riether et al., 2000). The phoA promoter from E. coli was fused to the promoterless luxCDABE operon in the pSA-derived vector pUCD615 (Rogowsky et al., 1987). This plasmid replicates in a variety of Gram-negative bacteria and has proven to be useful in bacterial constructs for the monitoring of environmental pollutants (Selifonova et al., 1993; Vollmer et al., 1997; Riether et al., 2001). An E. coli strain
carrying this plasmid-based phoA::luxCDABE transcriptional fusion was found to emit light in a dosedependent manner upon phosphate starvation. In this communication, we report on the construction and characterization of the above phosphate sensor system. In addition to E. coli, we used the more environmentally relevant Pseudomonas fluorescens DF57 strain (Kragelund et al., 1995) as a host for the reporter gene construct. The resultant sensor strains were tested for their ability to report biologically accessible concentrations of phosphate in both standard solutions and wastewater samples using a simple and rapid bioassay.
2. Materials and methods 2.1. Bacterial strains and culture conditions E. coli DH10B (Life Technologies) was used for cloning experiments and propagation of plasmids. This strain was also the host for plasmid pUCD607, which contains the luxCDABE genes from V. fischeri under the control of the tetracycline resistance promoter (Shaw and Kado, 1986) and confers a constitutive bioluminescence phenotype. E. coli MG1655 (wild type) and P. fluorescens DF57 (Kragelund et al., 1995) were the recipients for the reporter plasmid pPHO-lux, the resulting phosphate sensor strains were used in bioluminescence assays. E. coli strains were cultured aerobically at 37 jC in Luria Broth (LB) or in MOPS minimal medium (Neidhardt et al., 1974). P. fluorescens was grown aerobically at 28 jC in either LB or Davis Minimal Medium (DMM) (Neidhardt et al., 1974). When appropriate, media were supplemented with ampicillin (50 Ag/ml), kanamycin (20 Ag/ml), or chloramphenicol (20 Ag/ml). 2.2. Construction of plasmid pPHO-lux General procedures for strain and plasmid construction were performed according to Sambrook et al. (1989). The plasmid pPHO-lux containing a transcriptional fusion of the phoA gene to the luxCDABE operon was constructed in a manner similar to that previously described (Riether et al., 2001). The phoA promoter region of E. coli was amplified from strain MG1655 genomic DNA by PCR using modified
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upstream primer pPHO1 (5V-CCGGATCCTGACCGACGATACGGAG-3V) that created a BamH1 site (in italics sequence) at position 244 with respect to the phoA start codon and unmodified downstream primer pPHO2 (5V-GTCACCGAATTCAGTGAGGC3V) containing an EcoRI site at position + 720. Amplification was carried out with a PTC-100 thermocycler (MJ Research), with an initial denaturation step (94 jC for 5 min) followed by 30 cycles of 94 jC for 30 s, 55 jC for 45 s, and 72 jC for 1 min. A final extension was performed for 5 min at 72 jC. The resulting PCR product was digested with BamH1 and EcoRI and ligated into BamH1 – EcoRI-digested pUCD615 (Rogowsky et al., 1987), to yield plasmid pPHO-lux, in which the complete luxCDABE operon from V. fischeri was controlled by the phoA promoter. Plasmid pPHO-lux was introduced into E. coli MG1655 and P. fluorescens DF57 by electroporation (Dower et al., 1988; Itoh et al., 1994).
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2.3. Preparation of cells and luminescence measurements Overnight MOPS cultures of E. coli MG1655/ pPHO-lux were diluted to an OD600 of 0.1 in fresh MOPS and grown with shaking until the exponentially growing phase was reached (OD600 of 0.4 –0.6). The cells were harvested by centrifugation at 6000 g for 10 min at room temperature, washed twice with one volume of MOPS minimal medium without phosphate, and then suspended in the same medium to obtain a final OD600 of 10. The same protocol was applied to DMM cultures of P. fluorescens DF57/ pPHO-lux. The positive control E. coli DH10B/pUCD607 strain was prepared as follows: an overnight LB culture of the bacteria was harvested by centrifugation at OD600 of 3– 4, washed with one volume of 10-fold concentrated GGM medium (Riether et al., 2001). The
Fig. 1. Kinetics of phoA::luxCDABE induction as a function of phosphate concentration in E. coli/pPHO-lux (A) and P. fluorescens/pPHO-lux (B). Values are means F S.D. (n = 3).
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bacteria were then suspended in the same medium to an OD600 of 2 before the measurements. In the standard induction assay, 10 Al of freshly prepared cell suspension were added to 90 Al of test solution in opaque white 96-well microtiter plates (Dynatech, Germany). The plates were immediately placed in a temperature-controlled (26 jC) microtiter plate luminometer (LUMIstar, BMG LabTechnologies, Germany), and bioluminescence was recorded in intervals of 20 min for at least 3 h, each sample being measured for 10 s. Before each measurement cycle, the plates were shaken for 1 min in the luminometer. Raw luminescence values were presented in the instrument’s arbitrary relative light units (RLU). In some cases, we also calculated the induction coefficient, which is equal to the luminescence in a samplecontaining well, divided by that in well containing uninduced cells (that is, in the presence of 0.2 mM K2HPO4) at the same time point. The minimum detection threshold was determined as the concentration at which the induction coefficient was 2. For phosphate bioavailability experiments, results were expressed as the percentage of the luminescence measured in a control sample containing no phosphate or ligand. All experiments were conducted in triplicate and repeated at least two times. Luminescence in triplicates varied by less than 15%, whereas variations between experiments normally did not exceed 40%.
ison, total phosphorus and orthophosphate concentrations were determined in filtered samples according to standard methods (AFNOR NF T 90-023, 1983).
3. Results and discussion 3.1. Kinetics of phoA::luxCDABE induction in E. coli and P. fluorescens Representative luminescence patterns of E. coli/ pPHO-lux and P. fluorescens/pPHO-lux when incubated with variable amounts of orthophosphate are shown in Fig. 1. E. coli responded to phosphate deprivation by a dose-dependent increase in light emission measurable after only 40 min of incubation. However, because of the transient increase in background luminescence in the first part of the assay, extended incubation times gave higher induction coefficients. As a compromise between sensitivity
2.4. Wastewater samples Water samples (raw sewage and final effluent) were collected from a wastewater treatment plant in Metz, France. Suspended solids were removed by centrifugation (6000 g for 10 min), and the resulting supernatants were filtered through a 0.45-Am filter. These samples were diluted with distilled water to final concentrations of 5 – 75% of the original and then assayed for bioavailable phosphate according to the above standard induction assay. A control assay using E. coli DH10B/pUCD607 was conducted in parallel to assess sample toxicity and matrix effect. Results were presented as the percentage of luminescence relative to distilled water controls. Apparent bioavailable phosphate concentrations in water samples were calculated by comparing luminescence values in diluted samples with that of a standard calibration curve. For compar-
Fig. 2. Response of E. coli/pPHO-lux (A) and P. fluorescens/pPHOlux (B) to different phosphorus compounds. K2HPO4 ( y); tripolyphosphate (E); h-glycerophosphate (5); glucose-6-phosphate (o); pyrophosphate ( ). Values presented were measured after 140 and 120 min incubation for E. coli and P. fluorescens, respectively. Error bars represent the S.D. for n = 3.
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and response time, luminescence values measured after 140 min of induction were chosen for further experiments. At this time point, the detection threshold, defined as the minimal phosphate concentration causing at least a twofold increase over background luminescence, was 60 AM (see Fig. 2A). In P. fluorescens, the expression of the phoA::luxCDABE fusion was also dependent on external phosphate concentration, with maximal induction occurring after 120 min incubation. The phosphate detection threshold at this incubation time (40 AM; see Fig. 2B) was slightly lower than that estimated for E. coli. On the other hand, the induction pattern differed from that of E. coli by a lag before induction, lower luminescence intensity, and a stable baseline throughout the assay. The heterologous expression of the E. coli phoA promoter in P. fluorescens is not so surprising and is in agreement with previous observations suggesting similarities in the control of the PHO regulon between the two bacteria (Kragelund et al., 1995). Similar phosphate-dependent light emission was also ob-
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served when Acinetobacter sp. strain ADP1 was used as a host for pPHO-lux (not shown). 3.2. Analyte spectrum In addition to phosphate, we examined the response of our reporter strains when incubated with two inorganic phosphates (pyrophosphate and tripolyphosphate) and two organic phosphate esters (h-glycerophosphate and glucose-6-phosphate), which potentially serve as environmental phosphorus source for microorganisms. As shown in Fig. 2, all four compounds were able to inhibit light emission by the two reporter strains in a concentration-dependent manner. This indicated that they served to supply cells with phosphate, probably via breakdown by periplasmic phosphatases, and thus repressed the expression of the phoA::luxCDABE fusion. For E. coli, the detection thresholds of the tested compounds appeared to decrease in the following order: h-glycerophosphate>pyrophosphate>tripolyphosphate>glucose-6-phosphate. Even the latter
Fig. 3. Influence of different phosphate ligands on the response of E. coli/pPHO-lux (A, C) and P. fluorescens/pPHO-lux (B, D). Panels (A) and (B): inhibition of luminescence of the sensors strains by ligands in a phosphate-free medium. Panels (C) and (D): effect of increasing concentrations of ligands on the response of the sensor strains in an assay medium containing 100 AM K2HPO4. FeCl3 ( ); Al2(SO4)3 (n); LaCl3 (o). Luminescence values 140 and 120 min after induction were used for E. coli and P. fluorescens, respectively. Error bars represent the S.D. for n = 3.
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compound, however, repressed the phoA expression at concentrations 20-fold higher than that required with phosphate, indicating that this sensor strain primary reports the bioavailability of phosphate. Some substrates such as pyrophosphate and tripolyphosphate seemed to produce a biphasic response curve, with intermediate plateau values followed by a further reduction in the luminescence level. Since no specific transporter system has been reported for these compounds in E. coli (Wanner, 1996), they may enter the cell either through a nonspecific pathway or after hydrolyse by periplasmic phosphatase(s). Intermediate luminescence plateau values may therefore reflect the saturation of one transport/breakdown system followed by the setup of a second nonspecific one at higher substrate concentrations. When compared to E. coli, the response of P. fluorescens was characterized by smaller dynamic ranges for phoA induction, the absence of intermediate plateau for pyrophosphate and tripolyphosphate, and a strikingly high detection threshold for glucose-6-phosphate (20 mM). The latter findings illustrate the contrasting ecophysiology of the two reporter strains and probably reflect differences in their phosphatase activity profiles.
concentration of 100 AM K2HPO4, which is high enough to ensure a basal luminescence emission. Results were expressed as the percentage of the luminescence measured in a control sample containing no phosphate or ligand. Response values near 1 therefore indicated the absence of (bioavailable) phosphate. As expected, addition of these compounds reduced the bioavailability of phosphate to the two biosensor strains, as judged by the progressive increase in the luminescence signal (Fig. 3C and D). LaCl3 appeared to be the most effective phosphate ligand, a concentration of 75 AM LaCl3 (LaCl3/Pi ratios = 0.75) being sufficient to make phosphate totally unavailable to E. coli. For higher LaCl3/Pi ratios, however, the luminescence level decreased. Considering the results presented in Fig. 3A and B, this effect can probably be attributed to the toxicity of lanthanum ions. Similar patterns were observed for FeCl3 and Al2 (SO4)3, the induction level and subsequent lumines-
3.3. Phosphate bioavailability One key advantage of whole-cell biosensors over conventional methods for determination of environmental contaminants concentrations is their ability to assess the bioavailable or bio-active fraction of these compounds. To determine whether our sensor strains were able to distinguish available and immobilised forms of phosphate, bioassays were conducted in the presence of three known phosphate ligands: ferric and aluminium salts [FeCl3, Al2(SO4)3] that are commonly used in wastewater treatment to precipitate phosphorus and lanthanum (LaCl3), whose potency as alternative phosphate binder for the management of hyperphosphataemia has been evaluated (Zhu et al., 2002). To assess the potential toxicity of the ligands, the two reporter strains were exposed to increasing concentrations of these compounds in a phosphatefree medium (Fig. 3A, B). In both cases, lanthanum salts proved to be the most toxic ligand, followed by aluminium and iron salts. For bioavailibility experiment, varying concentrations of the phosphate ligands were added to an assay medium containing a fixed
Fig. 4. Luminescence response displayed by E. coli/pPHO-lux (o), P. fluorescens/pPHO-lux (E), and the control strain E. coli/ pUCD607 (n), to dilutions of wastewater samples from a treatment plant. Panel (A): influent. Panel (B): effluent. Values presented were measured after 140 and 120 min incubation for E. coli and P. fluorescens, respectively. Error bars represent the S.D. for n = 3.
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cence decrease being a balance between the effectiveness of phosphate complexation and toxicity of the ligands to each strain.
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was effectively removed in the treatment facility to reach concentrations far below the detection thresholds of the bioassay, as also confirmed by data obtained from chemical analysis (Table 1).
3.4. Application of the bioassay to analyse wastewater samples 4. Conclusions To test the usefulness of our reporter strains in more environmentally relevant samples, bioassays were performed on water samples collected from a wastewater treatment plant (Metz, France). Fig. 4 shows the bioluminescence of the two strains exposed to different dilutions of filtered raw sewage (influent) and treated effluent samples. A decrease in light production was observed with the two reporter strains when increasing influent concentrations (Fig. 4A). This suggested that influent sample contained sufficient phosphate to repress, at least in part, phoA expression in the reporter strains. Alternatively, the same decrease in light production could be the result of increase toxicity. To test this possibility, a control assay using E. coli DH10B/ pUCD607 was conducted in parallel. No significant reduction in luminescence was detected whatever the tested dilutions, ruling out toxicity and matrix effects associated with the influent sample. We therefore calculated the apparent bioavailable phosphate concentrations in influent for both E. coli/pPHO-lux and P. fluorescens/pPHO-lux, by comparing luminescence values in diluted sample with that of calibration curves. Results presented in Table 1 suggested that phosphate content of the raw sewage, as measured by chemical method, is only partially available to E. coli. By contrast, the phosphate level detected by P. fluorescens corresponded to that defined chemically. No dilution-dependent induction was observed with the effluent sample (Fig. 4B), indicating that phosphate Table 1 Phosphorus concentration in wastewater samples measured by the phosphate reporter strains or by standard chemical method Bioavailable phosphate (AM)
Chemically determined phosphorus (AM)
E. coli/ P. fluorescens/ Orthophosphate Total pPHO-lux pPHO-lux phosphorus Influent 110 F 30a 160 F 40a Effluent < 10b < 10b a b
161 0.2
Data represent the average F S.D. (n = 3). Lowest detection limit.
178 0.7
The above findings are consistent with those previously reported by Schreiter et al. (2001), whose CyanoSensor (a Synechococcus reporter strain carrying a chromosomal phoA::luxAB fusion) was shown to reflect the bioavailability of different phosphorus sources. The sensitivity of phosphate detection with our sensor strains is far lower than that reported for the CyanoSensor, thus precluding their use for the monitoring of environments with low phosphorus content, such as oligotrophic lakes or drinking water. However the plasmid-based reporter system used here provides the advantage to give information on phosphate concentration in a relatively short time, without the need for illumination as required for cyanobacterial cultures. In addition, since the phoA regulation mechanism appears to be functionally conserved in many bacteria, one might expect that introduction of the pPHO-lux plasmid in bacterial hosts with contrasting ecophysiology will generate new phosphate reporter strains. Depending upon their performance, one or more such strains can therefore be chosen for a specific application. For example, P. fluorescens appears better suited than E. coli for the semiquantitative measurement of phosphate in wastewaters and to control the efficiency of its removal in treatment plants. Even if chemical methods to determine phosphate are accurate and sensitive, they do not provide clear information on the availability of this element to organisms. The same is true for organic and inorganic phosphorus-containing compounds, whose bio-accessibility cannot be monitored in a real-time scale via chemical approaches. Our results from phosphate bioavailability experiment showed that the two reporter strains did not detect the portion of phosphate that is complexed with the ligands. Similarly, the accessibility of different phosphorus sources could be evaluated and, for some of them, appeared to vary between the two bacteria. In general, the data presented here clearly indicate the applicability of the phoA::lux concept to monitor phosphorus bioavailability. The assay is sim-
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ple, rapid and may be exploited for different purposes such as to assess the strength of new phosphatebinding compounds or to study conditions that affect phosphate and/or phosphorus-containing compounds’ bioavailability in environmental samples, in combination with positive control biosensors to rule out toxicity problems. Nevertheless, for such practical applications, the approach still requires some improvements. For example, plasmid instability and possible variations in plasmid copy number in the cells are factors that may alter the assay performance. One possibility to overcome this problem and provide a more stable system would be the integration of the phoA::lux fusion into the host strains chromosome. Future investigations will also address the lyophilisation or immobilisation of the reporter strains in polymeric matrix, which will limit luminescence variations between experiments and allow the design of an easyto-use test for bioavailable phosphate.
Acknowledgements We thank N. Babic for excellent technical assistance and P. Poupin for comments on this manuscript. The generous gifts of P. fluorescens strain DF57 by O. Nybroe, and plasmids pUCD615 and pUCD607 by C.I. Kado are acknowledged. Part of this work was supported by NANCIE and CIRSEE-Lyonnaise des Eaux.
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