Tactic responses to pollutants and their potential to increase biodegradation efficiency

Authors

  • J. Lacal,

    1. Department of Environmental Protection, Estación Experimental del Zaidín, Consejo Superior de Investigaciones Científicas, Granada, Spain
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  • J.A. Reyes-Darias,

    1. Department of Environmental Protection, Estación Experimental del Zaidín, Consejo Superior de Investigaciones Científicas, Granada, Spain
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  • C. García-Fontana,

    1. Department of Environmental Protection, Estación Experimental del Zaidín, Consejo Superior de Investigaciones Científicas, Granada, Spain
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  • J.-L. Ramos,

    1. Department of Environmental Protection, Estación Experimental del Zaidín, Consejo Superior de Investigaciones Científicas, Granada, Spain
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  • T. Krell

    Corresponding author
    • Department of Environmental Protection, Estación Experimental del Zaidín, Consejo Superior de Investigaciones Científicas, Granada, Spain
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Correspondence

Tino Krell, Estación Experimental del Zaidín, Consejo Superior de Investigaciones Científicas, C/Prof. Albareda 1, 18008 Granada, Spain. E-mail: tino.krell@eez.csic.es

Summary

A significant number of bacterial strains are able to use toxic aromatic hydrocarbons as carbon and energy sources. In a number of cases, the evolution of the corresponding degradation pathway was accompanied by the evolution of tactic behaviours either towards or away from these toxic carbon sources. Reports are reviewed which show that a chemoattraction to heterogeneously distributed aromatic pollutants increases the bioavailability of these compounds and their biodegradation efficiency. An extreme form of chemoattraction towards aromatic pollutants, termed ‘hyperchemotaxis’, was described for Pseudomonas putida DOT-T1E, which is based on the action of the plasmid-encoded McpT chemoreceptor. Cells with this phenotype were found of being able to approach and of establishing contact with undiluted crude oil samples. Although close McpT homologues are found on other degradation plasmids, the sequence of their ligand-binding domains does not share significant similarity with that of NahY, the other characterized chemoreceptor for aromatic hydrocarbons. This may suggest the existence of at least two families of chemoreceptors for aromatic pollutants. The use of receptor chimers comprising the ligand-binding region of McpT for biosensing purposes is discussed.

Introduction

Many bacteria show flagellum-mediated chemotactic behaviours. A chemotactic signalling pathway is initiated by an interaction of a given signal molecule with the chemoreceptor, and the resulting molecular stimulus is then transmitted to the CheA sensor kinase causing a modulation of its autokinase activity. Differences in the CheA phosphorylation state impact on the transphosphorylation activity to the CheY response regulator, of which its phosphorylated state binds to the flagellar motor thereby altering its activity. Other core proteins of a chemosensory cascade include the CheB and CheR methylesterase and methyltransferases, respectively, that mediate adaptive responses (Krell et al. 2011).

A major driving force in the evolution of chemotaxis is the capacity to sense and approach compounds that function as carbon or nitrogen sources (such as sugars, amino acids, Krebs cycle intermediates), or compounds that function as electron acceptors like oxygen, nitrate, fumarate, in the bacterial metabolism (Schweinitzer and Josenhans 2010). A significant number of chemoreceptors have been identified that mediate taxis towards these compounds as exemplified by the Tar (Mowbray and Koshland 1987), Tsr (Boyd et al. 1983), PctA, PctB, PctC (Taguchi et al. 1997), McpB (Glekas et al. 2010) and BasT (Kokoeva and Oesterhelt 2000) receptors for amino acids, Tar (Mowbray and Koshland 1987), Trg (Vyas et al. 1988) and McpA (Hanlon and Ordal 1994) for different sugars, McpS (Lacal et al. 2010a), PA2652 (Alvarez-Ortega and Harwood 2007) and TCP (Iwama et al. 2000) for Krebs cycle intermediates or Aer (Taylor 2007) and HemAT (Hou et al. 2000) for oxygen.

Free-living bacteria are frequently exposed to habitats in which the above carbon sources are scarce. The evolution of many of these bacteria resulted in a significant metabolic versatility that permits the use of a large number of additional carbon sources, such as aromatic hydrocarbons. This is exemplified by the evolution of at least six different degradation pathways that permit the metabolism of toluene (Parales et al. 2008). Aromatic hydrocarbons are toxic to life because they dissolve in the bacterial membrane leading to its disorganisation, which eventually leads to cell death (Ramos et al. 2002). To mediate the dangerous balance between the nutritious and toxic effects of these compounds, sophisticated and complex regulatory mechanisms are necessary. These mechanisms are frequently at the level of transcriptional regulation (Parales et al. 2008), but in many cases, specific chemotactic behaviours in response to these compounds have evolved (Parales et al. 2000). These responses are either chemoattraction or chemorepellation that leads either to an attraction of bacteria to a carbon source or, respectively, corresponds to an escape mechanism form toxic compounds (Shitashiro et al. 2005; Vardar et al. 2005).

We review studies that suggest that chemoattraction increases the bioavailability of heterogeneously distributed aromatic biodegradation substrates, which may render biodegradation processes more efficient. We emphasize on the hyperchemotaxis phenotype to aromatic hydrocarbons that is mediated by the McpT chemoreceptor. In addition, current information on aromatic hydrocarbon chemoreceptors is summarised and the use of chimeric proteins comprising the ligand-binding domain (LBD) of pollutant chemoreceptors for biosensor applications is discussed.

Tackling the limitations of biodegradation

Human activity is frequently associated with the generation of xenobiotics, which are compounds that are normally absent from the biosphere. Xenobiotics can interfere with central processes of life, causing a threat to life in general (Aitken et al. 2004). There are studies that show a direct link between the presence of xenobiotics and the reduction in human fertility (Oliva et al. 2001), whereas other reports show that xenobiotics increase the error rate during DNA duplication, altering the genomic patrimony of man (Aitken and De Iuliis 2007) or demonstrating a carcinogenic character (Clavel 2007).

Confronted with this threat, a polyphasic approach has been proposed to overcome this situation, which consists of (i) stringent regulations for the production and usage of toxic compounds, (ii) pretreatment and safer disposal of toxic compounds and (iii) restoration of contaminated sites (Pandey et al. 2009). Whereas the first two points are of preventive nature, bioremediation processes are a promising strategy to address the third point. Bioremediation processes are based on the exploitation of the metabolic potential of living organisms and are aimed at attenuating the toxic effects of pollutants. Soil and aquatic micro-organisms were found to be well suited for bioremediation purposes, because their natural habitat is frequently characterized by a shortage of nutrients, which has prompted the evolution of an enormous metabolic versatility (Timmis 2002). Research in the field of biodegradation has resulted in the development of a number of biodegradation methods. The application of these methods under in situ conditions has not yielded the expected results (Pandey et al. 2009) and was frequently found little efficient.

However, the limitations of the biodegradation processes have been clearly identified. The three major limitations can be summarised as follows: (i) insufficient resistance of the micro-organism when exposed to toxic compounds (Singer et al. 2005) (ii) insufficient expression of the enzymes of the degradation pathways (Cases and de Lorenzo 2005) and (iii) reduced bioavailability of the compound to be degraded (Head 1998).

Over the last decades, significant advances have been made in the understanding of the molecular mechanisms leading to limitations 1 and 2. It was shown that efflux pumps are central to guaranteeing bacterial solvent resistance (Rojas et al. 2001; Ramos et al. 2002). In addition, processes that alter the membrane composition were found to increase bacterial resistance, such as increased cis/trans isomerisation of unsaturated fatty acids (Heipieper et al. 2003), a change of the ratio of saturated to unsaturated fatty acids (Alsaker et al. 2010) or changes of the composition of phospholipid head groups (Zhao et al. 2003; Bernal et al. 2007; Pini et al. 2009).

Important insight has also been obtained as to reasons for the reduced expression of degradation pathways under in situ conditions. Several degradation pathways for aromatic solvents are controlled by two-component systems that belong to the family of TodS/TodT (Parales et al. 2008). It was shown that the sensor kinase TodS recognizes different mono- and biaromatic compounds, but gene induction was mediated by only a fraction of them, termed ‘agonists’ (Lacal et al. 2006). It was demonstrated that transcriptionally ineffective TodS ligands, termed ‘antagonists’, compete with agonists for binding to the same site on TodS (Busch et al. 2007), causing a reduction in agonist-mediated upregulation of transcription. The regulation of the homologous system TmoS/TmoT by the concerted action of agonists and antagonists has also been reported (Silva-Jimenez et al. 2012). Crude oil is a mixture of agonists and antagonists, and the magnitude of gene induction has to be considered as the results of the concerted action of agonistic and antagonistic compounds. Protein engineering of the corresponding sensor kinases aimed at repressing the binding or inhibitory action of antagonists may be a way to overcome this undesired property.

Chemotaxis of soil and aquatic bacteria: response to diverse types of pollutants

Different types of chemotaxis exist and in this article this term refers to flagellum-mediated taxis. Chemotaxis allows motile bacteria to migrate towards or away from different environmental signals. The core proteins of the chemotactic apparatus include the chemoreceptors, the histidine protein kinase CheA, the adaptor protein CheW, the response regulator CheY as well as the receptor-modification enzymes CheR and CheB (Wuichet and Zhulin 2010). Genome analyses reveal that more than half of the bacterial genomes possess chemotaxis genes (Wuichet and Zhulin 2010). In the past, the molecular mechanism of chemotaxis has been studied primarily in enterobacteria (Wadhams and Armitage 2004). The specificity of a chemotactic response is determined by the chemoreceptor that is typically composed of a periplasmatic LBD and a cytosolic signalling domain. Escherichia coli has four chemoreceptors that primarily mediate taxis towards some amino acids, sugars, dipeptides and pyrimidines (Hazelbauer et al. 2008; Liu and Parales 2008).

A genome analysis of all available genomes revealed that motile bacteria have on average 14 chemoreceptors (Lacal et al. 2010b), which is a value significantly above that for enterobacteria. The increased number of chemoreceptors in many free-living bacteria enables them to respond to a wider range of compounds as compared to enterobacteria (Lacal et al. 2010b; Krell et al. 2011). Interestingly, many of these compounds are chemicals of environmental concern. Chemoattraction was observed towards biphenyl, benzoic acid and chlorobenzoic acids (Tremaroli et al. 2010), toluene and its derivatives (Parales et al. 2000; Lacal et al. 2011), naphthalene and its derivatives (Grimm and Harwood 1997; Lacal et al. 2011), nitroaromatics (Iwaki et al. 2007), chloroaromatics (Gordillo et al. 2007), chloronitroaromatics (Pandey et al. 2012), aminoaromatics (Parales 2004), explosives (Leungsakul et al. 2005), aliphatic hydrocarbons (Lanfranconi et al. 2003) and herbicides (Liu and Parales 2009) in species like Rhizobium sp., Bradyrhizobium sp., Pseudomonas sp., Azospirillum sp., Ralstonia sp., Burkholderia sp. or Flavimonasoryzihabitans. In a significant number of cases, the physiological relevance of chemoattraction to pollutants lies in the fact that these compounds serve as carbon and energy sources. This may be exemplified by the chemotaxis towards toluene and naphthalene by Pseudomonas putida DOT-T1E and G7, which possess specific degradation routes for both compounds, respectively (Tsuda and Iino 1990; Mosqueda et al. 1999).

Many of the above-mentioned compounds serve as carbon sources, but are also toxic to life. It is therefore understandable why some bacteria have developed chemorepellent responses. Bacterial repellence has been reported, for example, to hydrogen peroxide, hypochlorite and N-chlorotaurine (Benov and Fridovich 1996), the PAHs anthracene and pyrene (Ortega-Calvo et al. 2003), Co++ and Ni++ (Tso and Adler 1974). Some chemicals can even be chemoattractants for one bacterial species and be repellent for another (Parales et al. 2000; Shitashiro et al. 2005; Vardar et al. 2005). The physical state of the chemical also appears to influence the type of response, because it was shown that the naphthalene degrader Ps. putida G7 was repelled by naphthalene in the vapour phase, whereas it was attracted when the compound was dissolved in the aqueous phase (Hanzel et al. 2010). In the light of such results, one has to keep in mind that an observed chemotaxis phenotype can be the result of the action of several, potential antagonistic chemoreceptors that differ in their sensitivity to a given compound.

Chemoattraction increases bioavailability of pollutants and enhances biodegradation rate

There is now evidence indicating that chemoattraction increases the bioavailability of heterogeneously distributed pollutants. In this context, the naphthalene taxis of the naphthalene degrader Ps. putida G7 may be a representative example. This strain contains a plasmid that encodes a naphthalene-degradation pathway and the NahY chemoreceptor that mediates chemotaxis towards this compound (Grimm and Harwood 1999). Interestingly, the NahY receptor and the meta cleavage degradation pathway were found to be cotranscribed (Grimm and Harwood 1999). The authors thus propose that the presence of the catabolic plasmid-encoded chemoreceptor might facilitate naphthalene biodegradation.

Proof of this hypothesis was brought by Aitken and co-workers. Using a heterogeneous aqueous system, they were able to demonstrate that chemotaxis enhances naphthalene biodegradation (Marx and Aitken 2000a). Subsequent studies using chemotactic and nonchemotactic strains of Ps. putida G7 clearly demonstrated that chemotaxis increased naphthalene degradation when the compound is present in a nonaqueous-phase-liquid (Law and Aitken 2003). The experimental data agreed with a mathematical model developed for the chemotaxis towards a consumable substrate in capillary assays (Marx and Aitken 2000b) and porous media (Pedit et al. 2002).

Bhushan et al. (2004) studied the biodegradation of cyclic nitramine explosives that are hydrophobic pollutants with very low water solubility. In sediment and soil environments, they are often attached to solid surfaces and/or trapped in pores and are distributed heterogeneously in aqueous environments. The authors were able to isolate the anaerobic bacterium Clostridium sp. strain EDB2, which was able to biotransform these compounds. Interestingly, biotransformation reactions that led to the production of NO2 caused a chemotactic behaviour which in turn was found to increase the biodegradation rate. In this case, it is not chemotaxis to the pollutant per se but to a derived metabolite, NO2, which stimulated biotransformation.

Another study revealed the link between chemotaxis and degradation of various nitroaromatic compounds (Samanta et al. 2000). Interestingly, chemotaxis was only observed towards compounds that were degraded by the micro-organism, whereas nonsubstrate compounds were not found to be chemoattractants. This confirms the link between chemotaxis and biodegradation. Another article reported the study of chemotaxis towards p-nitrophenol in soil using the chemotactic strain Ralstonia sp. and a nonchemotactic control (Paul et al. 2006). The authors successfully demonstrated chemotactic movement towards pollutants in a porous support such as soil. This work clearly supports the hypothesis that chemotaxis may enhance in situ bioremediation of toxic pollutants from soils and sediments.

Other studies have attributed a role for chemotaxis in directing bacterial migration towards contaminants in natural porous media under groundwater flow conditions (Wang and Ford 2009). Furthermore, deposition of chemotactic bacteria during transport in porous media was investigated (Velasco-Casal et al. 2008; Jimenez-Sanchez et al. 2012). These studies concluded that bacteria chemotactic to naphthalene or to salicylate (the latter an intermediate of naphthalene degradation) are deposited to a lesser extent in porous media than the control strain, which was devoid of chemotactic movement. Taken this information together, it can be concluded that there is a significant link between chemotaxis and the degradation of pollutants. This is consistent with the notion that an optimization of the chemotactic movement would lead to an increase in degradation efficiency.

Hyperchemotaxis towards pollutants

It has been reported that toluene degrading Ps. putida strains show a chemotactic behaviour towards this toxic aromatic compound (Parales et al. 2000). However, the corresponding chemoreceptor remained unidentified. In our laboratory, we have analysed the chemotactic response of several Ps. putida strains towards toluene using the agarose plug assay (Fig. 1) (Lacal et al. 2011). In this assay, the chemoattractant toluene was immobilised in an agarose plug, which was then exposed to a bacterial suspension. A ring formation close to the plug is indicative of chemotaxis. Agarose plugs containing buffer or the strong attractant succinate serve as negative and positive control, respectively (Fig. 1a). As for the taxis towards toluene, two different chemotaxis phenotypes were observed (Fig. 1a). For strains KT2440 and F1, ring formation occurred at a distance to the central plug and the corresponding phenotype was termed ‘moderate positive taxis’. Cells of strain DOT-T1E in contrast accumulated right on the surface of the toluene containing plug, which was referred to as hyperchemotaxis (Lacal et al. 2011). As shown in Fig. 1b, both phenotypes can also be clearly distinguished by an alternative assay in which the chemoattractant was placed into a capillary. In the case of hyperchemotaxis, cells accumulate right at the mouth of the capillary, whereas cells that show the moderate positive taxis accumulate at a distance. In contrast to the strains that exhibit moderate positive chemotaxis, strain DOT-T1E contains a large, self-transmissible plasmid termed pGRT1 (Rodriguez-Herva et al. 2007). To verify whether this plasmid might be responsible for the different phenotypes, the plasmid-free derivative DOT-T1E-100 was analysed. As shown in Fig. 1, this strain showed the moderate chemotaxis phenotype similar to the one observed for strains KT2440 and F1 (Lacal et al. 2011). When this plasmid was transferred to the KT2440 strain, the hyperchemotaxis phenotype was observed (Fig. 1) which proofs that the determinant for the hyperchemotaxis phenotype towards toluene is located on plasmid pGRT1. The sequence of this plasmid (Molina et al. 2011) has revealed the presence of two almost identical genes that encode a chemoreceptor which we have termed McpT. Mutation of one or the other mcpT allele was sufficient to revert the hyperchemotaxis phenotype into the moderate phenotype and a complementation of the mutant with a mcpT-containing plasmid restored the hyperchemotaxis phenotype (Fig. 2) (Lacal et al. 2011).

Figure 1.

Chemotactic behaviour of different strains of Pseudomonas putida towards toluene. (a) Agarose plug assays of strains F1, DOT-T1E and KT2440. Strain DOT-T1E-100 is a derivative of DOT-T1E lacking the large, self-transmissible plasmid pGRT1 (Rodriguez-Herva et al. 2007). Strain KT2440 (pGRT1::ΔttgV) has the ttgV gene disrupted by a kanamycin cassette. Strains presenting the hyperchemotaxis phenotype are underlined. Controls show the response of strain DOT-T1E to buffer and succinate. (b) Capillary assays of strains DOT-T1E, DOT-T1E-100 and KT2440 towards toluene that was immobilized in the capillary at a concentration of 10% (v/v). Reproduced with permission from (Lacal et al. 2011).

Figure 2.

The mcpT gene is responsible for the hyperchemotaxis phenotype. Shown are agarose plug assays of the wild-type strain Pseudomonas putida DOT-T1E, a strain with a mutated mcpT allele (Ps. putida DOT-T1E::ΔmcpT) and the latter strain complemented with a plasmid (pBBR1MCS-5::mcpT) harbouring the mcpT gene. Reproduced with permission from (Lacal et al. 2011).

Subsequent experiments revealed that the McpT chemoreceptor not only mediates a hyperchemotactic response to toluene but also to a wide range of different mono- and biaromatic compounds as well as to some aliphatic compounds (Lacal et al. 2011), of which most are not mineralized by the TOD pathway. The physiological sense of taxis towards compounds that are toxic but not mineralized is not understood. McpT is a transmembrane receptor containing a periplasmic LBD, predicted to form a four-helix bundle structure. A single helix-breaking point mutation in the final signalling helix of this domain resulted in receptor inactivation, that is compatible with the idea that ligands are sensed at the periplasmic LBD (Lacal et al. 2011). Interestingly, close homologues (99% sequence identity) of McpT are found on other catabolic plasmids of hydrocarbon degrading strains like pCAR1 of Pseudomonas resinovorans (Miyakoshi et al. 2007), the TOL plasmid pWW53 of Ps. putida (Yano et al. 2007) and the plasmid pMAQU02 of Marinobacter aquaeolei VT8, a strain isolated from the head of an oil well and able to degrade various crude oil components (Huu et al. 1999). This underlines the link between chemotaxis and the metabolism of aromatic hydrocarbons. However, in the case of McpT, there appears to be a link between the resistance and the chemotaxis towards aromatic hydrocarbons, because the genes of the TtgGHI efflux pump, the major determinant for the remarkable solvent resistance of this strain, are flanked by the two mcpT alleles (Molina et al. 2011).

The discovery of the McpT chemoreceptor is also of biotechnological relevance. One of the reasons for the lacking efficiency of biodegradation processes is the limited bioavailability of the target compounds (Head 1998). Chemotaxis has been shown in several cases to increase bioavailability of degradable compounds and consequently the efficiency of their biodegradation (Pandey et al. 2009). We have conducted chemotaxis assays of Ps. putida DOT-T1E and its mcpT mutant towards undiluted crude oil samples recovered from the Spanish coast following the ‘Prestige’ oil tanker accident. As shown in Fig. 3, the wild-type strain shows a pronounced movement towards this highly toxic mixture of compounds and accumulates on its surface, whereas the mcpT deficient mutant shows very moderate taxis and cells remain at safe distance to the crude oil sample.

Figure 3.

Chemotaxis of Pseudomonas putida DOT-T1E and its mutant devoid of both mcpT genes towards undiluted crude oil recovered from the Spanish coast following the ‘Prestige’ oil tanker accident. Reproduced with permission from (Lacal et al. 2011).

The plasmid pGRT1 contains two alleles for the mcpT chemoreceptor, but lacks genes for the cytosolic signalling proteins necessary for a chemotactic response. This implies that the hyperchemotaxis response is based on chemosensory pathways that involve plasmid-encoded chemoreceptors and genome-encoded signalling proteins. We have already shown that the transfer of the mcpT gene to other strains of the genus Ps. putida, such as strain KT2440 (Fig. 1), causes the hyperchemotactic phenotype indicative of the establishment of a functional chemosensory pathway using components from different strains. This offers the possibility of engineering hydrocarbon degrading strains by introducing chemoreceptor genes, which in turn is likely to promote bioavailability of aromatic hydrocarbons.

In summary, Ps. putida strains KT2440, F1 and the plasmid-free derivative of DOT-T1E show moderately positive chemotaxis. This taxis is mediated by (a) genome encoded chemoreceptor(s) that yet remain(s) to be identified. The plasmid-born chemoreceptor McpT induces an extreme form of taxis, termed ‘hyperchemotaxis’, towards a wide range of different hydrocarbons. The cellular compartment of signal detection is most likely the periplasm. The hyperchemotaxis phenotype is proposed to be an efficient means to increase the bioavailability of heterogeneously distributed biodegradation substrates.

Sequence divergence of identified pollutant chemoreceptors

The specificity of a chemotactic response is determined by chemoreceptors. Two chemoreceptors for aromatic pollutants have so far been described, which are NahY (Grimm and Harwood 1999) of the naphthalene degrading Ps. putida G7 and McpT of the toluene, benzene and ethylbenzene degrading Ps. putida DOT-T1E (Lacal et al. 2011). Both receptors mediate chemoattraction towards their respective degradation substrates. Interesting parallels exist between both receptors. NahY and McpT are encoded on plasmids pNAH7 (Sota et al. 2006) and pGRT1 (Molina et al. 2011), respectively. Both plasmids contain genes that are related to the degradation of or resistance to aromatic pollutants, respectively. Both receptors are predicted to contain two transmembrane regions that flank a periplasmic ligand-binding region (LBRs) of around 170 amino acids that classifies them as cluster I receptors. The secondary structure prediction of both LBRs is consistent with a four-helix bundle structure similar to that of the Tar receptor (Milburn et al. 1991). The specificity of ligand recognition of chemoreceptors and therefore their function is determined by the LBR. Although there is experimental evidence that McpT and NahY mediate taxis towards very similar compounds (i.e. both were found to respond to naphthalene), the sequence alignment of their LBRs showed only a nonsignificant identity of 15%. This similarity is in the same range than that observed between McpT-LBR and NahY-LBR (13–14%) with the LBR of Tar that binds and mediates responses towards aspartate. Therefore, a sequence-based identification of pollutant chemoreceptors is in many cases impossible, which is a major handicap in this field of research.

This relatively low sequence identity between sensor domains of pollutant chemoreceptors may also be in agreement with genetic and physiological data that show that chemotaxis to pollutants is characterized by a relatively low ligand specificity (Parales et al. 2000; Parales 2004; Gordillo et al. 2007). A low specificity of ligand recognition appears to be a binding mode that was frequently found for other proteins that recognize hydrophobic compounds such as efflux pumps (Eicher et al. 2009), one-component transcriptional regulators (Guazzaroni et al. 2005) and sensor kinases (Busch et al. 2007). To understand and exploit chemotaxis to pollutants, high-resolution structural information on the proteins involved is essential, which would in turn provide a rational basis to optimize this process by altering the receptor ligand profile or binding affinity.

Use of pollutant chemoreceptors for the construction of biosensors

Analytical techniques are necessary to detect and quantify environmental pollution. Many standard techniques use sophisticated equipment, but biosensors are interesting alternatives because they are generally cost-efficient and permit an easy use. Biosensors are constructed through the fusion of promoters, responsive to the relevant environmental conditions, to easily monitored reporter genes (Ron 2007). Recent advances in this area have included the development of biosensors of compact size that enable the on-line and in situ monitoring of a large number of environmental parameters (Ron 2007).

Many whole-cell biosensors for the detection of aromatic pollutants are based on the use of transcriptional regulators that recognize the target compounds (Fig. 4). In most cases, these transcriptional regulators control the expression of catabolic pathways. In this context, biosensors based on one-component systems XylR (Kim et al. 2005; Li et al. 2008; de Las Heras and de Lorenzo 2012), PhnR (Tecon et al. 2006), CapR (Shin et al. 2005) or NahR/NagR (Mitchell and Gu 2005; Kohlmeier et al. 2007) or two-component systems like StyS/StyR (Alonso et al. 2003) or TodS/TodT (Applegate et al. 1998; Dawson et al. 2008) have been described. In addition, biosensors have been developed which use transcriptional regulators that control pollutant efflux pumps such as in the case of the SepABC pump (Keane et al. 2008). However, the sensitivity of detection is a crucial parameter for any analytical technique. What all these above-mentioned sensors have in common is that the sensing occurs in the cytosol. Aromatic pollutants enter the cell by passive diffusion but accumulate in both cell membranes of Gram-negative bacteria (Ramos et al. 2002). Several efflux pumps have been shown to expulse aromatic pollutants from cell, which was found to be the primary mechanism of solvent tolerance (Rojas et al. 2001; Ramos et al. 2002). There are thus significant differences in the cellular and extracellular pollutant concentrations. This is supported by the observation that the cytosolic sensor kinase TodS binds toluene with a submicromolar dissociation constant, but extracellular concentrations of around 100 micromolar toluene are necessary to achieve half-maximal transcriptional activation (Lacal et al. 2006; Busch et al. 2007). The aim of new generation pollutant biosensors is to detect these compounds in the extracellular space. Due to the differences in cellular and extracellular pollutant concentration, biosensory systems based on extracellular detection have the promise of higher sensitivity.

Figure 4.

Different strategies for the development of biosensors for aromatic pollutants. Shown is the use of one-component systems, two-component systems and receptor chimera for biosensor application. The hexagon represents the aromatic compound, RR: response regulator.

Such systems can be developed by the construction of receptor chimera using pollutant chemoreceptor LBRs. The construction of chimera comprising the LBR of a chemosensor with the autokinase domain of a sensor kinase was successful in the case of the Taz chimera (Yoshida et al. 2007). This chimera comprises the periplasmic LBD of the Tar chemoreceptor fused to the autokinase domain of the EnvZ sensor kinase. Tar recognizes aspartate at its LBD and mediates chemotaxis towards this compound. The EnvZ/OmpR two-component system modulates the expression of porins in function of changes in osmolarity. Aspartate was shown to bind to the periplasmic LBD of Taz, which ultimately was found to upregulate porin expression (Jin and Inouye 1993). The functionality of Taz chimera and the identification of the McpT pollutant chemoreceptor form the basis for the construction of analogous chimera in which the LBR of McpT is fused to the autokinase domain of a transmembrane sensor kinase.

Acknowledgements

Work was supported by research grants from the Spanish Ministry of Science and Innovation (grants: BIO2010-16937, BIO2010-17227, CSD2007-0005), the Andalusian Regional Government Junta de Andalucía (research grants P09-RNM-4509 and group VVI-191) and the Fundación BBVA (research grant to T.K.).

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