Initially described in 1990, Pseudomonas fluorescens HK44 served as the first whole-cell bioreporter genetically endowed with a bioluminescent (luxCDABE) phenotype directly linked to a catabolic (naphthalene degradative) pathway. HK44 was the first genetically engineered microorganism to be released in the field to monitor bioremediation potential. Subsequent to that release, strain HK44 had been introduced into other solids (soils, sands), liquid (water, wastewater), and volatile environments. In these matrices, it has functioned as one of the best characterized chemically-responsive environmental bioreporters and as a model organism for understanding bacterial colonization and transport, cell immobilization strategies, and the kinetics of cellular bioluminescent emission. This review summarizes the characteristics of P. fluorescens HK44 and the extensive range of its applications with special focus on the monitoring of bioremediation processes and biosensing of environmental pollution.

Faculty of Environment Jan Evangelista Purkyně University in Ústí nad Labem Králova Výšina 3132 7 400 96 Ústí nad Labem Czech Republic

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Leveau J.H.J., Lindow S.E. Bioreporters in microbial ecology. Curr. Opin. Microbiol. 2002;5:259–265. PubMed

van der Meer J.R., Belkin S. Where microbiology meets microengineering: Design and applications of reporter bacteria. Nat. Rev. Microbiol. 2010;8:511–522. PubMed

Close D.M., Ripp S., Sayler G.S. Reporter proteins in whole-cell optical bioreporter detection systems, biosensor integrations, and biosensing applications. Sensors. 2009;9:9147–9174. PubMed PMC

King J.M.H., Digrazia P.M., Applegate B., Burlage R., Sanseverino J., Dunbar P., Larimer F., Sayler G.S. Rapid, sensitive bioluminescent reporter technology for naphthalene exposure and biodegradation. Science. 1990;249:778–781. PubMed

Burlage R.S., Sayler G.S., Larimer F. Monitoring of naphthalene catabolism by bioluminescence with nah-lux transcriptional fusions. J. Bacteriol. 1990;172:4749–4757. PubMed PMC

van der Meer J.R., Tropel D., Jaspers M. Illuminating the detection chain of bacterial bioreporters. Environ. Microbiol. 2004;6:1005–1020. PubMed

Sagi E., Hever N., Rosen R., Bartolome A.J., Premkumar J.R., Ulber R., Lev O., Scheper T., Belkin S. Fluorescence and bioluminescence reporter functions in genetically modified bacterial sensor strains. Sens. Actuat. B. 2003;90:2–8.

Diplock E.E., Alhadrami H.A., Paton G.I. Whole Cell Sensing Systems II. Vol. 118. Springer-Verlag; New York, NY, USA: 2010. Application of microbial bioreporters in environmental microbiology and bioremediation; pp. 189–209. PubMed

Sayler G.S., Fleming J.T., Nivens D.E. Gene expression monitoring in soils by mRNA analysis and gene lux fusions. Curr. Opin. Biotechnol. 2001;12:455–460. PubMed

Gu M., Mitchell R., Kim B. Biomanufacturing. Vol. 87. Springer; Berlin, Germany: 2004. Whole-cell-based biosensors for environmental biomonitoring and application; pp. 269–305. PubMed

Belkin S. Microbial whole-cell sensing systems of environmental pollutants. Curr. Opin. Microbiol. 2003;6:206–212. PubMed

Nunes-Halldorson V.D., Duran N.L. Bioluminescent bacteria: Lux genes as environmental biosensors. Braz. J. Microbiol. 2003;34:91–96.

Girotti S., Ferri E.N., Fumo M.G., Maiolini E. Monitoring of environmental pollutants by bioluminescent bacteria. Anal. Chim. Acta. 2008;608:2–29. PubMed

Sayler G.S., Ripp S. Field applications of genetically engineered microorganisms for bioremediation processes. Curr. Opin. Biotechnol. 2000;11:286–289. PubMed

Kohler S., Belkin S., Schmid R.D. Reporter gene bioassays in environmental analysis. Fresenius J. Anal. Chem. 2000;366:769–779. PubMed

Woutersen M., Belkin S., Brouwer B., van Wezel A.P., Heringa M.B. Are luminescent bacteria suitable for online detection and monitoring of toxic compounds in drinking water and its sources? Anal. Bioanal. Chem. 2011;400:915–929. PubMed PMC

Aller A.J., Castro M.A. Live bacterial cells as analytical tools for speciation analysis: Hypothetical or practical? TrAC Trends Anal. Chem. 2006;25:887–898.

Ripp S., DiClaudio M.L., Sayler G.S. Environmental Microbiology. 2nd ed. John Wiley & Sons, Inc; Hoboken, NJ, USA: 2010. Biosensors as environmental monitors; pp. 213–233.

Eltzov E., Marks R.S. Whole-cell aquatic biosensors. Anal. Bioanal. Chem. 2011;400:895–913. PubMed

Lei Y., Chen W., Mulchandani A. Microbial biosensors. Anal. Chim. Acta. 2006;568:200–210. PubMed

D’Souza S.F. Microbial biosensors. Biosens. Bioelectron. 2001;16:337–353. PubMed

Su L.A., Jia W.Z., Hou C.J., Lei Y. Microbial biosensors: A review. Biosens. Bioelectron. 2011;26:1788–1799. PubMed

Simpson M.L., Sayler G.S., Applegate B.M., Ripp S., Nivens D.E., Paulus M.J., Jellison G.E. Bioluminescent-bioreporter integrated circuits form novel whole-cell biosensors. Trends Biotechnol. 1998;16:332–338.

Meighen E.A. Genetics of bacterial bioluminescence. Annu. Rev. Genet. 1994;28:117–139. PubMed

Meighen E.A. Bacterial bioluminescence—Organization, regulation, and application of the lux genes. FASEB J. 1993;7:1016–1022. PubMed

Meighen E.A., Dunlap P.V. Advances in Microbial Physiology. Vol. 34. Academic Press; Waltham, MA, USA: 1993. Physiological, biochemical and genetic-control of bacterial bioluminescence; pp. 1–67. PubMed

Meighen E.A. Molecular-biology of bacterial bioluminescence. Microbiol. Rev. 1991;55:123–142. PubMed PMC

Meighen E.A. Enzymes and genes from the lux operons of bioluminescent bacteria. Annu. Rev. Microbiol. 1988;42:151–176.

Samanta S.K., Singh O.V., Jain R.K. Polycyclic aromatic hydrocarbons: Environmental pollution and bioremediation. Trends Biotechnol. 2002;20:243–248. PubMed

Peng R.H., Xiong A.S., Xue Y., Fu X.Y., Gao F., Zhao W., Tian Y.S., Yao Q.H. Microbial biodegradation of polyaromatic hydrocarbons. FEMS Microbiol. Rev. 2008;32:927–955. PubMed

Yen K.M., Serdar C.M. Genetics of naphthalene catabolism in pseudomonads. Crit. Rev. Microbiol. 1988;15:247–268. PubMed

Yen K.M., Gunsalus I.C. Plasmid gene organization—Naphthalene salicylate oxidation. Proc. Natl. Acad. Sci. USA. 1982;79:874–878. PubMed PMC

Diaz E., Prieto M.A. Bacterial promoters triggering biodegradation of aromatic pollutants. Curr. Opin. Biotechnol. 2000;11:467–475. PubMed

Juwarkar A.A., Singh S.K., Mudhoo A. A comprehensive overview of elements in bioremediation. Rev. Environ. Sci. Biotechnol. 2010;9:215–288.

Caliman F.A., Robu B.M., Smaranda C., Pavel V.L., Gavrilescu M. Soil and groundwater cleanup: Benefits and limits of emerging technologies. Clean Technol. Environ. Policy. 2011;13:241–268.

Singh J.S., Abhilash P.C., Singh H.B., Singh R.P., Singh D.P. Genetically engineered bacteria: An emerging tool for environmental remediation and future research perspectives. Gene. 2011;480:1–9. PubMed

Pandey J., Chauhan A., Jain R.K. Integrative approaches for assessing the ecological sustainability of in situ bioremediation. FEMS Microbiol. Rev. 2009;33:324–375. PubMed

Kuncová G., Trögl J. Physiology of microorganisms encapsulated into inorganic polymers. In: Morrison D.A., editor. Handbook of Inorganic Chemistry Research. Nova Science Publishers; New York, NY, USA: 2010. pp. 53–101.

Webb O.F., Bienkowski P.R., Matrubutham U., Evans F.A., Heitzer A., Sayler G.S. Kinetics and response of a Pseudomonas fluorescens HK44 biosensor. Biotechnol. Bioeng. 1997;54:491–502. PubMed

Engebrecht J., Nealson K., Silverman M. Bacterial bioluminescence—Isolation and genetic-analysis of functions from Vibrio fischeri. Cell. 1983;32:773–781. PubMed

Ørskov I., Ørskov F. Plasmid-determined H2S character in Escherichia coli and its relation to plasmid-carried raffinose fermentation and tetracycline resistance characters. J. Gen. Microbiol. 1973;77:487–499. PubMed

Chauhan A., Layton A.C., Williams D.E., Smartt A.E., Ripp S., Karpinets T.V., Brown S.D., Sayler G.S. Draft genome sequence of the polycyclic aromatic hydrocarbon-degrading, genetically engineered bioluminescent bioreporter Pseudomonas fluorescens HK44. J. Bacteriol. 2011;193:5009–5010. PubMed PMC

Kamath R., Schnoor J.L., Alvarez P.J.J. Effect of root-derived substrates on the expression of nah-lux genes in Pseudomonas fluorescens HK44: Implications for PAH biodegradation in the rhizosphere. Environ. Sci. Technol. 2004;38:1740–1745. PubMed

Dennis J.J., Zylstra G.J. Complete sequence and genetic organization of pDTG1, the 83 kilobase naphthalene degradation plasmid from Pseudomonas putida strain NCIB 9816-4. J. Mol. Biol. 2004;341:753–768. PubMed

Heinaru E., Vedler E., Jutkina J., Aava M., Heinaru A. Conjugal transfer and mobilization capacity of the completely sequenced naphthalene plasmid pNAH20 from multiplasmid strain Pseudomonas fluorescens PC20. FEMS Microbiol. Ecol. 2009;70:563–574. PubMed

Schell M.A. Transcriptional control of the nah and sal hydrocarbon-degradation operons by the NahR gene-product. Gene. 1985;36:301–309. PubMed

Schell M.A. Molecular-biology of the LysR family of transcriptional regulators. Annu. Rev. Microbiol. 1993;47:597–626. PubMed

Heitzer A., Webb O.F., Thonnard J.E., Sayler G.S. Specific and quantitative assessment of naphthalene and salicylate bioavailability by using a bioluminescent catabolic reporter bacterium. Appl. Environ. Microbiol. 1992;58:1839–1846. PubMed PMC

Schell M.A., Wender P.E. Identification of the NahR gene-product and nucleotide-sequences required for its activation of the sal operon. J. Bacteriol. 1986;166:9–14. PubMed PMC

Kelly C.J., Hsiung C.J., Lajoie C.A. Kinetic analysis of bacterial bioluminescence. Biotechnol. Bioeng. 2003;81:370–378. PubMed

Heitzer A., Malachowsky K., Thonnard J.E., Bienkowski P.R., White D.C., Sayler G.S. Optical biosensor for environmental online monitoring of naphthalene and salicylate bioavailability with an immobilized bioluminescent catabolic reporter bacterium. Appl. Environ. Microbiol. 1994;60:1487–1494. PubMed PMC

Trogl J., Ripp S., Kuncova G., Sayler G., Churava A., Parik P., Demnerova K., Halova J., Kubicova L. Selectivity of whole cell optical biosensor with immobilized bioreporter Pseudomonas fluorescens HK44. Sens. Actuat. B. 2005;107:98–103.

Matrubutham U., Thonnard J.E., Sayler G.S. Bioluminescence induction response and survival of the bioreporter bacterium Pseudomonas fluorescens HK44 in nutrient-deprived conditions. Appl. Microbiol. Biotechnol. 1997;47:604–609.

Heitzer A., Applegate B., Kehrmeyer S., Pinkart H., Webb O.F., Phelps T.J., White D.C., Sayler G.S. Physiological considerations of environmental applications of lux reporter fusions. J. Microbiol. Methods. 1998;33:45–57.

Trogl J., Kuncova G., Kubicova L., Parik P., Halova J., Demnerova K., Ripp S., Sayler G. Response of the bioluminescent bioreporter Pseudomonas fluorescens HK44 to analogs of naphthalene and salicylic acid. Folia Microbiol. 2007;52:3–14. PubMed

Trogl J., Halova J., Kuncova G., Parik P. Automatic formation of hypotheses on the relationships between structure of naphthalene analogs and bioluminescence response of bioreporter Pseudomonas fluorescens HK44. Folia Microbiol. 2010;55:411–417. PubMed

Trogl J., Kuncova G., Kuran P. Bioluminescence of Pseudomonas fluorescens HK44 in the course of encapsulation into silica gel. Effect of methanol. Folia Microbiol. 2010;55:569–575. PubMed

Bundy J.G., Campbell C.D., Paton G.I. Comparison of response of six different luminescent bacterial bioassays to bioremediation of five contrasting oils. J. Environ. Monit. 2001;3:404–410. PubMed

Bundy J.G., Maciel H., Cronin M.T.D., Paton G.I. Limitations of a cosolvent for ecotoxicity testing of hydrophobic compounds. Bull. Environ. Contam. Toxicol. 2003;70:1–8. PubMed

Burlage R.S., Palumbo A.V., Heitzer A., Sayler G. Bioluminescent reporter bacteria detect contaminants in soil samples. Appl. Biochem. Biotechnol. 1994;45–46:731–740.

Ford C.Z., Sayler G.S., Burlage R.S. Containment of a genetically engineered microorganism during a field bioremediation application. Appl. Microbiol. Biotechnol. 1999;51:397–400. PubMed

Ripp S., Nivens D.E., Ahn Y., Werner C., Jarrell J., Easter J.P., Cox C.D., Burlage R.S., Sayler G.S. Controlled field release of a bioluminescent genetically engineered microorganism for bioremediation process monitoring and control. Environ. Sci. Technol. 2000;34:846–853.

Ripp S., Nivens D.E., Werner C., Sayler G.S. Bioluminescent most-probable-number monitoring of a genetically engineered bacterium during a long-term contained field release. Appl. Microbiol. Biotechnol. 2000;53:736–741. PubMed

Uesugi S.L., Yarwood R.R., Selker J.S., Bottomley P.J. A model that uses the induction phase of lux gene-dependent bioluminescence in Pseudomonas fluorescens HK44 to quantify cell density in translucent porous media. J. Microbiol. Methods. 2001;47:315–322. PubMed

Bundy J.G., Morriss A.W.J., Durham D.G., Campbell C.D., Paton G.I. Development of QSARs to investigate the bacterial toxicity and biotransformation potential of aromatic heterocylic compounds. Chemosphere. 2001;42:885–892. PubMed

Ripp S., Nivens D.E., Werner C., Sayler G.S. Vertical transport of a field-released genetically engineered microorganism through soil. Soil Biol. Biochem. 2001;33:1873–1877.

Yarwood R.R., Rockhold M.L., Niemet M.R., Selker J.S., Bottomley P.J. Noninvasive quantitative measurement of bacterial growth in porous media under unsaturated-flow conditions. Appl. Environ. Microbiol. 2002;68:3597–3605. PubMed PMC

Rockhold M.L., Yarwood R.R., Niemet M.R., Bottomley P.J., Selker J.S. Considerations for modeling bacterial-induced changes in hydraulic properties of variably saturated porous media. Adv. Water Resour. 2002;25:477–495.

O’Neill S., Ripp S., Megginson C., Davies I.M. Microbial Detection of Polycyclic Aromatic Hydrocarbons in Contaminated Marine Sediment. Fisheries Research Services; Aberdeen, UK: 2003.

Kuncova G., Podrazky O., Ripp S., Trogl J., Sayler G., Demnerova K., Vankova R. Monitoring of the viability of cells immobilized by sol-gel process. J. Sol-Gel Sci. Technol. 2004;31:335–342.

Valdman E., Valdman B., Battaglini F., Leite S.G.F. On-line detection of low naphthalene concentrations with a bioluminescent sensor. Process Biochem. 2004;39:1217–1222.

Valdman E., Battaglini F., Leite S.G.F., Valdman B. Naphthalene detection by a bioluminescence sensor applied to wastewater samples. Sens. Actuat. B. 2004;103:7–12.

Patterson C.J., Semple K.T., Paton G.I. Non-exhaustive extraction techniques (NEETs) for the prediction of naphthalene mineralisation in soil. FEMS Microbiol. Lett. 2004;241:215–220. PubMed

Rockhold M.L., Yarwood R.R., Niemet M.R., Bottomley P.J., Selker J.S. Experimental observations and numerical modeling of coupled microbial and transport processes in variably saturated sand. Vadose Zone J. 2005;4:407–417.

Yarwood R.R., Rockhold M.L., Niemet M.R., Selker J.S., Bottomley P.J. Impact of microbial growth on water flow and solute transport in unsaturated porous media. Water Resour. Res. 2006 doi: 10.1029/2005WR004550. DOI

Silva T.R., Valdman E., Valdman B., Leite S.G.F. Salicylic acid degradation from aqueous solutions using Pseudomonas fluorescens HK44: Parameters studies and application tools. Braz. J. Microbiol. 2007;38:39–44.

Rockhold M.L., Yarwood R.R., Niemet M.R., Bottomley P.J., Brockman F.J., Selker J.S. Visualization and modeling of the colonization dynamics of a bioluminescent bacterium in variably saturated, translucent quartz sand. Adv. Water Resour. 2007;30:1593–1607.

Valdman E., Gutz I.G.R. Bioluminescent sensor for naphthalene in air: Cell immobilization and evaluation with a dynamic standard atmosphere generator. Sens. Actuat. B. 2008;133:656–663.

Paton G.I., Reid B.J., Sempled K.T. Application of a luminescence-based biosensor for assessing naphthalene biodegradation in soils from a manufactured gas plant. Environ. Pollut. 2009;157:1643–1648. PubMed

Diplock E.E., Mardlin D.P., Killham K.S., Paton G.I. Predicting bioremediation of hydrocarbons: Laboratory to field scale. Environ. Pollut. 2009;157:1831–1840. PubMed

Kuncova G., Pazlarova J., Hlavata A., Ripp S., Sayler G.S. Bioluminescent bioreporter Pseudomonas putida TVA8 as a detector of water pollution. Operational conditions and selectivity of free cells sensor. Ecol. Indic. 2011;11:882–887.

LeBlond J.D., Applegate B.M., Menn F.M., Schultz T.W., Sayler G.S. Structure-toxicity assessment of metabolites of the aerobic bacterial transformation of substituted naphthalenes. Environ. Toxicol. Chem. 2000;19:1235–1246.

Sayre P. Risk Assessment for a recombinant biosensor. In: Sayler G.S., Sanseverino J., Davis K.L., editors. Biotechnology in the Sustainable Environment. 1st ed. Plenum Press; New York, NY, USA: 1997. pp. 269–279.

Junter G.A., Jouenne T. Immobilized viable microbial cells: From the process to the proteome. or the cart before the horse. Biotechnol. Adv. 2004;22:633–658. PubMed

Oates P.M., Castenson C., Harvey C.F., Polz M., Culligan P. Illuminating reactive microbial transport in saturated porous media: Demonstration of a visualization method and conceptual transport model. J. Contam. Hydrol. 2005;77:233–245. PubMed

Menn F.M., Applegate B.M., Sayler G.S. Nah plasmid-mediated catabolism of anthracene and phenanthrene to naphthoic acids. Appl. Environ. Microbiol. 1993;59:1938–1942. PubMed PMC

Sanseverino J., Applegate B.M., King J.M.H., Sayler G.S. Plasmid-mediated mineralization of naphthalene, phenanthrene, and anthracene. Appl. Environ. Microbiol. 1993;59:1931–1937. PubMed PMC

Nivens D.E., McKnight T.E., Moser S.A., Osbourn S.J., Simpson M.L., Sayler G.S. Bioluminescent bioreporter integrated circuits: Potentially small, rugged and inexpensive whole-cell biosensors for remote environmental monitoring. J. Appl. Microbiol. 2004;96:33–46. PubMed

Vijayaraghavan R., Islam S.K., Zhang M., Ripp S., Caylor S., Bull N.D., Moser S., Terry S.C., Blalock B.J., Sayler G.S. A bioreporter bioluminescent integrated circuit for very low-level chemical sensing in both gas and liquid environments. Sens. Actuat. B. 2007;123:922–928.

Islam S.K., Vijayaraghavan R., Zhang M., Ripp S., Caylor S.D., Weathers B., Moser S., Terry S., Blalock B.J., Sayler G.S. Integrated circuit biosensors using living whole-cell bioreporters. IEEE Trans. Circuits Syst. I-Regul. Pap. 2007;54:89–98.

Bolton E.K., Sayler G.S., Nivens D.E., Rochelle J.M., Ripp S., Simpson M.L. Integrated CMOS photodetectors and signal processing for very low-level chemical sensing with the bioluminescent bioreporter integrated circuit. Sens. Actuat. B. 2002;85:179–185. PubMed

del Busto-Ramos M., Budzik M., Corvalan C., Morgan M., Turco R., Nivens D., Applegate B. Development of an online biosensor for in situ monitoring of chlorine dioxide gas disinfection efficacy. Appl. Microbiol. Biotechnol. 2008;78:573–580. PubMed

Cook K.L., Garland J.L., Layton A.C., Dionisi H.M., Levine L.H., Sayler G.S. Effect of microbial species richness on community stability and community function in a model plant-based wastewater processing system. Microb. Ecol. 2006;52:725–737. PubMed

Mogil’naya O.A., Krivomazova E.S., Kargatova T.V., Lobova T.I., Popova L.Y. Formation of structured communities by natural and transgenic naphthalene-degrading bacteria. Appl. Biochem. Microbiol. 2005;41:63–68. PubMed

Ho C.H., Applegate B., Banks M.K. Impact of microbial/plant interactions on the transformation of polycyclic aromatic hydrocarbons in rhizosphere of Festuca arundinacea. Int. J. Phytorem. 2007;9:107–114. PubMed

de Weger L.A., Kuiper I., van der Bij A.J., Lugtenberg B.J.J. Use of a lux-based procedure to rapidly visualize root colonisation by Pseudomonas fluorescens in the wheat rhizosphere. Antonie Van Leeuwenhoek. 1997;72:365–372. PubMed

Nguy D., Sedgley C. The influence of canal curvature on the mechanical efficacy of root canal irrigation in vitro using real-time imaging of bioluminescent bacteria. J. Endod. 2006;32:1077–1080. PubMed

Sedgley C.M., Nagel A.C., Hall D., Applegate B. Influence of irrigant needle depth in removing bioluminescent bacteria inoculated into instrumented root canals using real-time imaging in vitro. Int. Endod. J. 2005;38:97–104. PubMed

Falk K.W., Sedgley C.M. The influence of preparation size on the mechanical efficacy of root canal irrigation in vitro. J. Endod. 2005;31:742–745. PubMed

Sedgley C., Applegate B., Nagel A., Hall D. Real-time imaging and quantification of bioluminescent bacteria in root canals in vitro. J. Endod. 2004;30:893–898. PubMed

Elad T., Benovich E., Magrisso S., Belkin S. Toxicant identification by a luminescent bacterial bioreporter panel: Application of pattern classification algorithms. Environ. Sci. Technol. 2008;42:8486–8491. PubMed

Jouanneau S., Durand M.J., Courcoux P., Blusseau T., Thouand G. Improvement of the identification of four heavy metals in environmental samples by using predictive decision tree models coupled with a set of five bioluminescent bacteria. Environ. Sci. Technol. 2011;45:2925–2931. PubMed

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