Experimental Evolution of Novel Regulatory Activities in Response to Hydrocarbons and Related Chemicals

  • V. ShinglerEmail author
Reference work entry
Part of the Handbook of Hydrocarbon and Lipid Microbiology book series (HHLM)


Bacterial transcriptional regulatory proteins that control catabolism of hydrocarbons and related chemicals have evolved (or are actively evolving) toward specifically detecting compounds that signal the presence of growth substrates. Laboratory evolution of the chemical-binding and response properties of sensory regulators has been achieved by a number of different techniques to generate novel derivatives with desired properties. Such manipulated and selected regulatory proteins are increasingly used in artificial genetic circuitry for improved biodegradation systems, biosensor construction, and in assembling regulatory cascades for synthetic biology within a wide range of biotechnological applications.



Research in the Shingler laboratory is supported by the Swedish Research Council (VR-MH 2016-02047) and the JC Kempe and SM Kempe foundation (JCK-1523).


  1. Beggah S, Vogne C, Zenaro E, van de Meer JR (2008) Mutant HbpR transcription activator isolation for 2-chlorobiphenyl via green flourescent protein-based flow cytometry and cell sorting. Microb Biotechnol 1:68–78PubMedGoogle Scholar
  2. Cases I, de Lorenzo V (2005) Promoters in the environment: transcriptional regulation in its natural context. Nat Rev Microbiol 3:105–118CrossRefGoogle Scholar
  3. Cebolla A, Sousa C, de Lorenzo V (1997) Effector specificity mutants of the transcriptional activator NahR of naphthalene degrading Pseudomonas define protein sites involved in binding of aromatic inducers. J Biol Chem 272:3986–3992CrossRefGoogle Scholar
  4. Cebolla A, Sousa C, de Lorenzo V (2001) Rational design of a bacterial transcriptional cascade for amplifying gene expression capacity. Nucleic Acids Res 29:759–766CrossRefGoogle Scholar
  5. Collier DN, Spence C, Cox MJ, Phibbs PV (2001) Isolation and phenotypic characterization of Pseudomonas aeruginosa pseudorevertants containing suppressors of the catabolite repression control-defective crc-10 allele. FEMS Microbiol Lett 196:87–92CrossRefGoogle Scholar
  6. de Las HA, de Carreno CA, de Lorenzo V (2008) Stable implantation of orthogonal sensor circuits in Gram-negative bacteria for environmental release. Environ Microbiol 10:3305–3316CrossRefGoogle Scholar
  7. Delgado A, Ramos JL (1994) Genetic evidence for activation of the positive transcriptional regulator Xy1R, a member of the NtrC family of regulators, by effector binding. J Biol Chem 269:8059–8062PubMedGoogle Scholar
  8. Devesse L, Smirnova I, Lonneborg R, Kapp U, Brzezinski P, Leonard GA, Dian C (2011) Crystal structures of DntR inducer binding domains in complex with salicylate offer insights into the activation of LysR-type transcriptional regulators. Mol Microbiol 81:354–367CrossRefGoogle Scholar
  9. Dominguez-Cuevas P, Marin P, Busby S, Ramos JL, Marques S (2008) Roles of effectors in XylS-dependent transcription activation: intramolecular domain derepression and DNA binding. J Bacteriol 190:3118–3128CrossRefGoogle Scholar
  10. Galvao TC, de Lorenzo V (2006) Transcriptional regulators a la carte: engineering new effector specificities in bacterial regulatory proteins. Curr Opin Biotechnol 17:34–42CrossRefGoogle Scholar
  11. Galvao TC, Mencia M, de Lorenzo V (2007) Emergence of novel functions in transcriptional regulators by regression to stem protein types. Mol Microbiol 65:907–919CrossRefGoogle Scholar
  12. Garmendia J, Devos D, Valencia A, de Lorenzo V (2001) A la carte transcriptional regulators: unlocking responses of the prokaryotic enhancer-binding protein XylR to non-natural effectors. Mol Microbiol 42:47–59CrossRefGoogle Scholar
  13. Gupta S, Saxena M, Saini N, Mahmooduzzafar, Kumar R, Kumar A (2012) An effective strategy for a whole-cell biosensor based on putative effector interaction site of the regulatory DmpR protein. PLOS ONE 7:e43527CrossRefGoogle Scholar
  14. Kwon HJ, Bennik MH, Demple B, Ellenberger T (2000) Crystal structure of the Escherichia coli Rob transcription factor in complex with DNA. Nat Struct Biol 7:424–430CrossRefGoogle Scholar
  15. Lonneborg R, Smirnova I, Dian C, Leonard GA, Brzezinski P (2007) In vivo and in vitro investigation of transcriptional regulation by DntR. J Mol Biol 372:571–582CrossRefGoogle Scholar
  16. Lonneborg R, Varga E, Brzezinski P (2012) Directed evolution of the transcriptional regulator DntR: isolation of mutants with improved DNT-response. PLoS One 7:e29994CrossRefGoogle Scholar
  17. Looger LL, Dwyer MA, Smith JJ, Hellinga HW (2003) Computational design of receptor and sensor proteins with novel functions. Nature 423:185–190CrossRefGoogle Scholar
  18. Maddocks SE, Oyston PC (2008) Structure and function of the LysR-type transcriptional regulator (LTTR) family proteins. Microbiology 154:3609–3623CrossRefGoogle Scholar
  19. Mahr R, Frunzke J (2016) Transcription factor-based biosensors in biotechnology: current state and future prospects. Appl Microbiol Biotechnol 100:79–90CrossRefGoogle Scholar
  20. Michan C, Zhou L, Gallegos MT, Timmis KN, Ramos JL (1992) Identification of critical amino-terminal regions of XylS. The positive regulator encoded by the TOL plasmid. J Biol Chem 267:22897–22901PubMedGoogle Scholar
  21. Mohn WW, Garmendia J, Galvao TC, de Lorenzo V (2006) Surveying biotransformations with a la carte genetic traps: translating dehydrochlorination of lindane (gamma-hexachlorocyclohexane) into lacZ-based phenotypes. Environ Microbiol 8:546–555CrossRefGoogle Scholar
  22. Ng LC, O’Neill E, Shingler V (1996) Genetic evidence for interdomain regulation of the phenol-responsive final σ 54-dependent activator DmpR. J Biol Chem 271:17281–17286CrossRefGoogle Scholar
  23. O’Neill E, Sze CC, Shingler V (1999) Novel effector control through modulation of a preexisting binding site of the aromatic-responsive σ 54-dependent regulator DmpR. J Biol Chem 274:32425–32432CrossRefGoogle Scholar
  24. O’Neill E, Wikstrom P, Shingler V (2001) An active role for a structured B-linker in effector control of the σ 54-dependent regulator DmpR. EMBO J 20:819–827CrossRefGoogle Scholar
  25. Park HH, Lee HY, Lim WK, Shin HJ (2005) NahR: effects of replacements at Asn 169 and Arg 248 on promoter binding and inducer recognition. Arch Biochem Biophys 434:67–74CrossRefGoogle Scholar
  26. Patil VV, Park KH, Lee SG, Woo E (2016) Structural analysis of the phenol-responsive sensory domain of the transcription activator PoxR. Structure 24:624–630CrossRefGoogle Scholar
  27. Pavel H, Forsman M, Shingler V (1994) An aromatic effector specificity mutant of the transcriptional regulator DmpR overcomes the growth constraints of Pseudomonas sp. strain CF600 on para-substituted methylphenols. J Bacteriol 176:7550–7557CrossRefGoogle Scholar
  28. Ramos JL, Stolz A, Reineke W, Timmis KN (1986) Altered effector specificities in regulators of gene expression: TOL plasmid xylS mutants and their use to engineer expansion of the range of aromatics degraded by bacteria. Proc Natl Acad Sci USA 83:8467–8471CrossRefGoogle Scholar
  29. Reimer A, Yagur-Kroll S, Belkin S, Roy S, van der Meer JR (2014) Escherichia coli ribose binding protein based bioreporters revisited. Sci Rep 4:5626CrossRefGoogle Scholar
  30. Rhee S, Martin RG, Rosner JL, Davies DR (1998) A novel DNA-binding motif in MarA: the first structure for an AraC family transcriptional activator. Proc Natl Acad Sci USA 95:10413–10418CrossRefGoogle Scholar
  31. Royo JL, Becker PD, Camacho EM, Cebolla A, Link C, Santero E, Guzman CA (2007) In vivo gene regulation in Salmonella spp. by a salicylate-dependent control circuit. Nat Methods 4:937–942CrossRefGoogle Scholar
  32. Sarand I, Skarfstad E, Forsman M, Romantschuk M, Shingler V (2001) Role of the DmpR-mediated regulatory circuit in bacterial biodegradation properties in methylphenol-amended soils. Appl Environ Microbiol 67:162–171CrossRefGoogle Scholar
  33. Schleif R (2003) AraC protein: a love-hate relationship. BioEssays 25:274–282CrossRefGoogle Scholar
  34. Shingler V (2003) Integrated regulation in response to aromatic compounds: from signal sensing to attractive behaviour. Environ Microbiol 5:1226–1241CrossRefGoogle Scholar
  35. Shingler V (2011) Signal sensory systems that impact σ54-dependent transcription. FEMS Microbiol Rev 35:425–440CrossRefGoogle Scholar
  36. Silva-Rocha R, de Lorenzo V (2008) Mining logic gates in prokaryotic transcriptional regulation networks. FEBS Lett 582:1237–1244CrossRefGoogle Scholar
  37. Skärfstad E, O’Neill E, Garmendia J, Shingler V (2000) Identification of an effector specificity subregion within the aromatic-responsive regulators DmpR and XylR by DNA shuffling. J Bacteriol 182:3008–3016CrossRefGoogle Scholar
  38. Stagno JR, Liu Y, Bhandari YR, Conrad CE, Panja S, Swain M, Fan L, Nelson G, Li C, Wendel DR, White TA, Coe JD, Wiedorn MO, Knoska J, Oberthuer D, Tuckey RA, Yu P, Dyba M, Tarasov SG, Weierstall U et al (2016) Structures of riboswitch RNA reaction states by mix-and-inject XFEL serial crystallography. Nature. doi:10.1038/nature20599CrossRefPubMedPubMedCentralGoogle Scholar
  39. Tropel D, van der Meer JR (2004) Bacterial transcriptional regulators for degradation pathways of aromatic compounds. Microbiol Mol Biol Rev 68:474–500CrossRefGoogle Scholar
  40. Uchiyama T, Abe T, Ikemura T, Watanabe K (2005) Substrate-induced gene-expression screening of environmental metagenome libraries for isolation of catabolic genes. Nat Biotechnol 23:88–93CrossRefGoogle Scholar
  41. van der Meer JR, Belkin S (2010) Where microbiology meets microengineering: design and applications of reporter bacteria. Nat Rev Microbiol 8:511–522CrossRefGoogle Scholar
  42. van Sint FS, Beilen JB, van Witholt B (2006) Selection of biocatalysts for chemical synthesis. Proc Natl Acad Sci USA 103:1693–1698CrossRefGoogle Scholar
  43. Wikström P, O’Neill E, Ng LC, Shingler V (2001) The regulatory N-terminal region of the aromatic-responsive transcriptional activator DmpR constrains nucleotide-triggered multimerisation. J Mol Biol 314:971–984CrossRefGoogle Scholar
  44. Wise AA, Kuske CR (2000) Generation of novel bacterial regulatory proteins that detect priority pollutant phenols. Appl Environ Microbiol 66:163–169CrossRefGoogle Scholar
  45. Zhang N, Darbari VC, Glyde R, Zhang X, Buck M (2016) The bacterial enhancer-dependent RNA polymerase. Biochem J 473:3741–3753CrossRefGoogle Scholar

Copyright information

© Springer Nature Switzerland AG 2019

Authors and Affiliations

  1. 1.Department of Molecular BiologyUmeå UniversityUmeåSweden

Personalised recommendations