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Upgrading bioluminescent bacterial bioreporter performance by splitting the lux operon

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Abstract

Bioluminescent bacterial bioreporters harbor a fusion of bacterial bioluminescence genes (luxCDABE), acting as the reporting element, to a stress-response promoter, serving as the sensing element. Upon exposure to conditions that activate the promoter, such as an environmental stress or the presence of an inducing chemical, the promoter::reporter fusion generates a dose-dependent bioluminescent signal. In order to improve bioluminescent bioreporter performance we have split the luxCDABE genes of Photorhabdus luminescens into two smaller functional units: luxAB, that encode for the luciferase enzyme, which catalyzes the luminescence reaction, and luxCDE that encode for the enzymatic complex responsible for synthesis of the reaction’s substrate, a long-chain aldehyde. The expression of each subunit was put under the control of either an inducible stress-responsive promoter or a synthetic constitutive promoter, and different combinations of the two units were tested for their response to selected chemicals in Escherichia coli. In all cases tested, the split combinations proved to be superior to the native luxCDABE configuration, suggesting an improved efficiency in the transcription and/or translation of two small gene units instead of a larger one with the same genes. The best combination was that of an inducible luxAB and a constitutive luxCDE, indicating that aldehyde availability is limited when the five genes are expressed together in E. coli, and demonstrating that improved biosensor performance may be achieved by rearrangement of the lux operon genes.

Splitting the Photorhabdus luminescens luxCDABE genes into two independently controlled units in E. coli dramatically enhaced microbial bioreporter performance

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References

  1. Harms H, Wells M, van der Meer J (2006) Whole-cell living biosensors-are they ready for environmental application? Appl Microbiol Biotechnol 70(3):273–280

    Article  CAS  Google Scholar 

  2. Rodriguez-Mozaz S, Lopez de Alda M, Barceló D (2006) Biosensors as useful tools for environmental analysis and monitoring. Anal Bioanal Chem 386(4):1025–1041

    Article  CAS  Google Scholar 

  3. van der Meer JR, Belkin S (2010) Where microbiology meets microengineering: design and applications of reporter bacteria. Nat Rev Microbiol 8(7):511–522

    Article  Google Scholar 

  4. Belkin S (2003) Microbial whole-cell sensing systems of environmental pollutants. Curr Opin Microbiol 6(3):206–212

    Article  CAS  Google Scholar 

  5. Sørensen S, Burmølle M, Hansen L (2006) Making bio-sense of toxicity: new developments in whole-cell biosensors. Curr Opin Biotechnol 17(1):11–16

    Article  Google Scholar 

  6. Tecon R, van der Meer J (2008) Bacterial biosensors for measuring availability of environmental pollutants. Sensors 8:4062–4080

    Article  CAS  Google Scholar 

  7. Stocker J, Balluch D, Gsell M, Harms H, Feliciano J, Daunert S, Malik K, vdM JR (2003) Development of a set of simple bacterial biosensors for quantitative and rapid measurements of arsenite and arsenate in potable water. Environ Sci Technol 37(20):4743–4750. doi:10.1021/es034258b

    Article  CAS  Google Scholar 

  8. Yagur-Kroll S, Bilic B, Belkin S (2010) Strategies for enhancing bioluminescent bacterial sensor performance by promoter region manipulation. Microb Biotechnol 3(3):300–310. doi:10.1111/j.1751-7915.2009.00149.x

    Article  CAS  Google Scholar 

  9. Davidov Y, Rozen R, Smulski D, Van Dyk T, Vollmer A, Elsemore D, LaRossa R, Belkin S (2000) Improved bacterial SOS promoter::lux fusions for genotoxicity detection. Mutat Res Genet Toxicol Environ Mutagen 466(1):97–107

    Article  CAS  Google Scholar 

  10. Maehana K, Tani H, Sa T, Kamidate T (2004) Effects of using a low-copy plasmid and controlling membrane permeability in SOS-based genotoxic bioassay. Anal Chim Acta 522(2):189–195

    Article  CAS  Google Scholar 

  11. Shapiro E, Baneyx F (2002) Stress-based identification and classification of antibacterial agents: second-generation Escherichia coli reporter strains and optimization of detection. Antimicrob Agents Chemother 46(8):2490–2497. doi:10.1128/aac.46.8.2490-2497.2002

    Article  CAS  Google Scholar 

  12. Meighen E (1991) Molecular biology of bacterial bioluminescence. Microbiol Mol Biol Rev 55(1):123–142

    CAS  Google Scholar 

  13. Craney A, Hohenauer T, Xu Y, Navani NK, Li Y, Nodwell J (2007) A synthetic luxCDABE gene cluster optimized for expression in high-GC bacteria. Nucl Acids Res 35(6):e46. doi:10.1093/nar/gkm086

    Article  Google Scholar 

  14. Qazi SNA, Counil E, Morrissey J, Rees CED, Cockayne A, Winzer K, Chan WC, Williams P, Hill PJ (2001) Agr expression precedes escape of internalized Staphylococcus aureus from the host endosome. Infect Immun 69(11):7074–7082. doi:10.1128/iai.69.11.7074-7082.2001

    Article  CAS  Google Scholar 

  15. Beard SJ, Salisbury V, Lewis RJ, Sharpe JA, MacGowan AP (2002) Expression of lux genes in a clinical isolate of Streptococcus pneumoniae: using bioluminescence to monitor gemifloxacin activity. Antimicrob Agents Chemother 46(2):538–542. doi:10.1128/aac.46.2.538-542.2002

    Article  CAS  Google Scholar 

  16. Francis KP, Joh D, Bellinger-Kawahara C, Hawkinson MJ, Purchio TF, Contag PR (2000) Monitoring bioluminescent Staphylococcus aureus infections in living mice using a novel luxCDABE construct. Infect Immun 68(6):3594–3600. doi:10.1128/iai.68.6.3594-3600.2000

    Article  CAS  Google Scholar 

  17. Mesak LR, Yim G, Davies J (2009) Improved lux reporters for use in Staphylococcus aureus. Plasmid 61(3):182–187

    Article  CAS  Google Scholar 

  18. Manen D, Pougeon M, Damay P, Geiselmann J (1997) A sensitive reporter gene system using bacterial luciferase based on a series of plasmid cloning vectors compatible with derivatives of pbr322. Gene 186(2):197–200

    Article  CAS  Google Scholar 

  19. Allen M, Wilgus J, Chewning C, Sayler G, Simpson M (2007) A destabilized bacterial luciferase for dynamic gene expression studies. Syst Synth Biol 1(1):3–9

    Article  Google Scholar 

  20. Hu J, Sauer R, Newell N, Tidor B (1993) Probing the roles of residues at the e and g positions of the gcn4 leucine zipper by combinatorial mutagenesis. Protein Sci 2(7):1072–1084

    Article  CAS  Google Scholar 

  21. Menzel R (1989) A microtiter plate-based system for the semiautomated growth and assay of bacterial cells for [beta]-galactosidase activity. Anal Biochem 181(1):40–50

    Article  CAS  Google Scholar 

  22. Jensen P, Hammer K (1998) The sequence of spacers between the consensus sequences modulates the strength of prokaryotic promoters. Appl Microbiol Biotechnol 64(1):82–87

    CAS  Google Scholar 

  23. Belkin S, Smulski D, Dadon S, Vollmer A, Van Dyk T, Larossa R (1997) A panel of stress-responsive luminous bacteria for the detection of selected classes of toxicants. Water Res 31(12):3009–3016

    Article  CAS  Google Scholar 

  24. Belkin S (1998) A panel of stress-responsive luminous bacteria for monitoring wastewater toxicity. Methods Mol Biol: Bioluminescent Protocols 102 (Humana, Totowa, NJ):247–258

  25. Quillardet P, Frelat G, Nguyen V, Hofnung M (1989) Detection of ionizing radiations with the SOS Chromotest, a bacterial short-term test for genotoxic agents. Mutat Res 216(5):251–257

    CAS  Google Scholar 

  26. Quillardet P, Huisman O, D’Ari R, Hofnung M (1982) SOS Chromotest, a direct assay of induction of an SOS function in Escherichia coli k-12 to measure genotoxicity. Proc Natl Acad Sci USA 79(19):5971–5975

    Article  CAS  Google Scholar 

  27. Nakamura S, Oda Y, Shimada T, Oki I, Sugimoto K (1987) SOS-inducing activity of chemical carcinogens and mutagens in Salmonella typhimurium ta1535/psk1002: examination with 151 chemicals. Mutat Res 192(4):239–246

    Article  CAS  Google Scholar 

  28. Belkin S, Smulski D, Vollmer A, Van Dyk T, LaRossa R (1996) Oxidative stress detection with Escherichia coli harboring a katG’::lux fusion. Appl Environ Biotechnol 62(7):2252–2256

    CAS  Google Scholar 

  29. Lee H, Gu M (2003) Construction of a sodA::luxCDABE fusion Escherichia coli: comparison with a katG fusion strain through their responses to oxidative stresses. Appl Microbiol Biotechnol 60(5):577–580

    CAS  Google Scholar 

  30. Ramanathan S, Shi W, Rosen B, Daunert S (1998) Bacteria-based chemiluminescence sensing system using [beta]-galactosidase under the control of the ArsR regulatory protein of the ars operon. Anal Chim Acta 369(3):189–195

    Article  CAS  Google Scholar 

  31. Oh J-T, Cajal Y, Skowronska EM, Belkin S, Chen J, Van Dyk T, Sasser M, Jain M (2000) Cationic peptide antimicrobials induce selective transcription of micF and osmY in Escherichia coli. Biochim Biophys Acta - Biomembranes 1463(1):43–54

    Article  CAS  Google Scholar 

  32. Fernández-Pinas F, Wolk CP (1994) Expression of luxCD-E in Anabaena sp. can replace the use of exogenous aldehyde for in vivo localization of transcription by luxAB. Gene 150(1):169–174

    Article  Google Scholar 

  33. Miyamoto C, Boylan M, Graham A, Meighen E (1988) Organization of the lux structural genes of Vibrio harveyi. Expression under the T7 bacteriophage promoter, mRNA analysis, and nucleotide sequence of the luxD gene. J Biol Chem 263(26):13393–13399

    CAS  Google Scholar 

  34. Welham P, Stekel D (2009) Mathematical model of the lux luminescence system in the terrestrial bacterium Photorhabdus luminescens. Mol Biosyst 5(1):68–76

    Article  CAS  Google Scholar 

  35. Philp J, French C, Wiles S, Bell J, Whiteley A, Bailey M (2004) Wastewater toxicity assessment by whole cell biosensor. The Handbook of Environmental Chemistry 5, Part I:165–225

  36. Rowe L, Combs K, Deo S, Ensor C, Daunert S, Qu X (2008) Genetically modified semisynthetic bioluminescent photoprotein variants: simultaneous dual-analyte assay in a single well employing time resolution of decay kinetics. Anal Chem 80(22):8470–8476

    Article  CAS  Google Scholar 

Download references

Acknowledgments

Research was supported by EU 6th framework project Toxichip (http://www.toxichip.org) and by BARD grant number US-3864-06. We are grateful to O. Amster-Choder, The Hebrew University, Jerusalem, Israel and to R. A. LaRossa, DuPont Company, Wilmington, Delaware, USA for the gift of E. coli strains AG1688 and RFM443, respectively.

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Authors and Affiliations

  1. Institute of Life Sciences, The Hebrew University of Jerusalem, Jerusalem, 91904, Israel

    Sharon Yagur-Kroll & Shimshon Belkin

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  1. Sharon Yagur-Kroll
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  2. Shimshon Belkin
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Correspondence to Shimshon Belkin.

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Published in the special issue Microorganisms for Analysis with Guest Editor Gérald Thouand.

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Yagur-Kroll, S., Belkin, S. Upgrading bioluminescent bacterial bioreporter performance by splitting the lux operon. Anal Bioanal Chem 400, 1071–1082 (2011). https://doi.org/10.1007/s00216-010-4266-7

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  • DOI: https://doi.org/10.1007/s00216-010-4266-7

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