Whole-Cell Fluorescent Bio Sensors for PCB Det

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Sensors 2010, 10, 1377-1398; doi:10.3390/s100201377 OPEN ACCESS sensors ISSN 1424-8220 www.mdpi.com/journal/sensors Review Whole-Cell Fluorescent Biosensors for Bioavailability and Biodegradation of Polychlorinated Biphenyls Xuemei Liu, Kieran J. Germaine, David Ryan and David N. Dowling * Department of Science and Health, Institute of Technology Carlow, Kilkenny Road, Carlow, Ireland; E-Mails: Liux@itcarlow.ie (X.M.L.); germaink@itcarlow.ie (K.J.G.); david.ryan@itcarlow.ie (D.R.) * Author to
  Sensors   2010 , 10 , 1377-1398; doi:10.3390/s100201377  sensors ISSN 1424-8220 www.mdpi.com/journal/sensors  Review  Whole-Cell Fluorescent Biosensors for Bioavailability andBiodegradation of Polychlorinated Biphenyls Xuemei Liu, Kieran J. Germaine, David Ryan and David N. Dowling * Department of Science and Health, Institute of Technology Carlow, Kilkenny Road, Carlow, Ireland;E-Mails: Liux@itcarlow.ie (X.M.L.);germaink@itcarlow.ie(K.J.G.);david.ryan@itcarlow.ie(D.R.) * Author to whom correspondence should be addressed; E-Mail: david.dowling@itcarlow.ie;Tel.: +353-59-91-75507; Fax: +353-59-9175501.  Received: 29 December 2009; in revised form: 14 January 2010 / Accepted: 29 January 2010 / Published: 21 February 2010 Abstract: Whole-cell microbial biosensors are one of the newest molecular tools used inenvironmental monitoring. Such biosensors are constructed through fusing a reporter genesuch as lux , gfp or lacZ  ,   to a responsive promoter. There have been many reports of theapplications of biosensors, particularly their use in assaying pollutant toxicity andbioavailability. This paper reviews the basic concepts behind the construction of whole-cellmicrobial biosensors for pollutant monitoring, and describes the applications of two suchbiosensors for detecting the bioavailability and biodegradation of PolychlorinatedBiphenyls (PCBs). Keywords: biosensor; Pseudomonas F113; PCB; biodegradation 1.   Introduction Environmental risk assessment is an essential tool in the investigation of polluted sites. Monitoringpractices for assessing these risks usually involve the determination of the total concentration of pollutants using sophisticated chemical analytical techniques such as Gas Chromatography-MassSpectroscopy (GC-MS) or High Performance Liquid Chromatography (HPLC) assays. The use of thetotal concentration is likely to overestimate the risk as only a fraction of the total amount of thepollutant, the bioavailable fraction, will actually have an impact on living organisms; this inability todifferentiate between the two represents a major disadvantage of traditional analytical methods. Thisdiscrepancy between the total and the bioavailable fractions is particularly significant in the case of  OPEN ACCESS   Sensors 2010 , 10   1378 contaminants with poor aqueous solubility (e.g., PCBs, Poly Aromatic Hydrocarbons [PAHs]) [1]. Theability to monitor the bioavailability of a pollutant is essential, as it not only gives more accurateinformation regarding the risk that the contaminated site poses to human health, but also determinesthe effectiveness of potential bioremediation processes. Nowadays, increasing attention has been givento bioavailability   assays that better predict the real exposure risks [2]. One such alternative is the use of biosensors which are highly selective and sensitive to a particular pollutant.Whole-cell microbial biosensors have become one of the newest dimensions of molecular tools inenvironmental monitoring [3-5]. Microorganisms, due to their low cost, lifespan, and range of suitablepH and temperatures, have been widely employed as the biosensing elements in the construction of biosensors [6].In the past decade, their applications were mainly focused in three areas:    Monitoring survival and competition ability of bacteria [7-11].    Monitoring plant root colonization of pollutant degrading bacteria in complex environmentalsamples [10,12-14].    Monitoring the level of specific environmental pollutants [13,15-20].In recent years, one of the most interesting areas utilising biosensor technology is the detection of environmental pollutant bioavailability, bioremediation, and toxicity. These biosensors are constructedby fusing a pollutant-responsive promoter to a reporter gene coding for a protein that can be easilyquantified, and such constructs can be located on plasmids or on the chromosome (Figure 1). Theefficacy of such biosensors was demonstrated by Willardson et al. [21]. The results they obtainedshowed that their toluene sensing, luciferase based whole-cell biosensor accurately reported tolueneconcentrations that were within the ±3% range as measured by standard GC-MS. Figure 1. Graphic illustrating the concept of a whole-cell transcriptional biosensor.The biosensors rely on analysis of gene expression, typically by creating transcriptional fusionsbetween a promoter of interest and the reporter gene. The extent of reporter gene expression may serveas a measure of the availability of specific pollutants in complex environments. Novel areas forapplying these biosensors have been previously documented and include the construction of whole-cellbiosensors as specific and sensitive devices for measuring biologically relevant concentrations of pollutants [4,15-18,21-27].  Sensors 2010 , 10   1379 Previous applications of whole-cell microbial biosensors for environmental studies mainlyconcentrated on their use as biomarkers to investigate survival and competition ability [7-11] and asbiosensors to detect the bioavailability or toxicity of environmental pollutants [15,16,28-33]. Layton et al . [34] reported a bioluminescent biosensor strain,  Ralstonia eutropha ENV307 (pUTK60), detectingthe bioavailability of PCBs by inserting the biphenyl promoter upstream of the bioluminescence genes.In the presence of biphenyl, bioluminescence was generated in a concentration-dependent manner.Kohler et al . [35] used an immobilized recombinant  E. coli reporter to detect the bioavailability of 4-chlorobenzoate.Compared with traditional detection methods for monitoring environmental pollutants, whole-cellbiosensors provide the following advantages [36]:    Biosensors determine only the bioavailable fraction of compounds, thus giving a more accurateresponse on the toxicity of a sample. Bioavailability is also important in bioremediation. If substances are bioavailable, they are potentially biodegradable.    Biosensors provide an inexpensive and simple way of determining contaminants.    As they are living organisms, they provide information on toxicology of different compounds.    Some stress-induced biosensors report the mutagenic effects of samples with great sensitivity.    Biosensors are unsurpassed in studying gene expression and physiology of bacteria in complexenvironments. 1.1. Commonly Used Reporter Genes The reporter gene usually encodes an enzyme catalyzing a reaction that can be easily monitored. Itdetermines the sensitivity and detection limits of the biosensor. Specific characteristics are needed forthe reporter gene to be used in a biosensor. The gene must have an expression or activity that can bemeasured using a simple assay and it must reflect the level of chemical or physical change. Also, thebiosensor must be free of any gene expression   or activity similar to the desired gene expression oractivity that is being measured to prevent misinterpretation of the response [37]. Several reporter genesmeet the necessary requirements and are frequently used including gfp , lacZ  , lucFF  , luxAB , and luxCDABE  with gfp and luxCDABE  [29,38-40] being the most commonly used.The gfp gene encoding Green Fluorescent Protein (GFP), srcinated from the jellyfish  Aequoreavictoria and its chromophore is assembled by the self-catalyzed covalent modification of amino acidsSer-Tyr-Gly at positions 65-67 to form a  p -hydroxybenzylidene-imidazolidinone species [41,42]. Thewild-type chromophore is excited with UV or blue light at 396 nm or 475 nm and emits greenfluorescence at 508 nm [41]. The fluorescence of GFP can be monitored without the destruction of thebiological sample [42,43]. A large collection of GFP derivatives have been constructed by the optimizationof codon usage to alter the spectral properties of GFP for use in different organisms [41,44,45]. There aremany examples of using different derivatives of GFPs in the construction of microbial biosensors fordetecting environmental pollutants [46-50].The bioluminescence gene lux cloned from Vibrio fischeri , Photorhabdus luminescens andothers [51], coding for the enzyme luciferase, is another reporter gene regularly used for theconstruction of biosensors to monitor environmental pollutants. The light emitted by the labeled straincan be proportional to the concentration of the target pollutant. Bioluminescence has been used very  Sensors 2010 , 10   1380 successfully as a reporter for pollutant detection using sensitive instrumentation including fiber opticprobes and integrated circuit chips detecting light production [52,53]. A comprehensive review of theapplication of bioluminescent genes and bacteria from 2000-2007 was reported by Girotti et al . [54].Li et al . [26] constructed toluene bacterial biosensors which comprised of two reporters, gfp and luxCDABE  . The bacterial luminescence biosensor allowed faster and more sensitive detection of toluene, while the fluorescence biosensor strain was much more stable and thus more applicable forlong-term exposure. 1.2. Promoters and Regulatory Elements for the Construction of Biosensors The selection of the promoter portion of the biosensor construct is dependent on the target molecule  being monitored. A selected promoter sequence is normally placed at the 5′ -region of the reportersystem where it can be switched on in the presence of the target pollutant, thus turning on theexpression of the reporter. The key factors when choosing promoters are sensitivity and specificity.Promoters often respond to groups of compounds rather than to a specific compound, and may alsobehave differently in different microorganisms. e.g., Winther-Larsen et al . [55] stated that theexpression of the p m promoter is substrate-dependent and host-specific (more details on this promoterare described in 2.1).A variety of well-characterized promoters are available for the construction of pollutant-reportingbiosensors. These promoters include those for hydrocarbons and organic solvents [56-60], variousheavy metals [17,18,61-63], pesticides [64,65], salicylates [66], various organo-phosphorous nerveagents [67,68], and mutagens and genotoxins [69,70]. Promoters are also available for the evaluationof general toxicity [71-74].One of the greatest limitations of whole-cell biosensor development is the availability of strongpromoters that respond only to relevant stimuli. To circumvent this obstacle, more knowledge on generegulatory networks in bacteria is needed. Linking metagenome information with the meta-transcriptome analysis of microbial communities using microarray technology could provide animmense source of new regulatory elements in the future [75] . Another option is to synthesize „super  promoters‟ based on consensus sequences obtained from comparative studies of different promoters in known regulatory networks [75]. 2. Development of Biosensors to Detect PCB Biodegradation PCBs were detected in the environment for the first time in 1966 by Jensen [76], and they havesince been found all over the world including in Arctic and Antarctic regions [77]. The production of PCBs was banned in 1970 in the USA and in the Czech Republic in 1984 [78]. However, severalhundred million kilograms has been released into the environment. Wiegel and Wu [79] documentedthat one-third of all US produced PCBs currently reside in the natural environment.One of the major threats to public health from PCBs is that they accumulate within the foodchain [80,81]. Contaminated fish consumption is a major route of PCB bioaccumulation inhumans [82]. The bioaccumulation capability of PCBs in salmon has increased to a much higher extentthan in other foods [83]. Traditional methods applied in the remediation of PCB contamination include
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