Increasing Signal Specificity of the TOL Network of mt-2 by Rewiring the Connectivity of the Master Regulator XylR

English version České info

Prokaryotic transcription factors (TFs) that bind small xenobiotic molecules (e.g., TFs that drive genes that respond to environmental pollutants) often display a promiscuous effector profile for analogs of the bona fide chemical signals. XylR, the master TF for expression of the m-xylene biodegradation operons encoded in the TOL plasmid pWW0 of Pseudomonas putida, responds not only to the aromatic compound but also, albeit to a lesser extent, to many other aromatic compounds, such as 3-methylbenzylalcohol (3MBA). We have examined whether such a relaxed regulatory scenario can be reshaped into a high-capacity/high-specificity regime by changing the connectivity of this effector-sensing TF within the rest of the circuit rather than modifying XylR structure itself. To this end, the natural negative feedback loop that operates on xylR transcription was modified with a translational attenuator that brings down the response to 3MBA while maintaining the transcriptional output induced by m-xylene (as measured with a luxCDABE reporter system). XylR expression was then subject to a positive feedback loop in which the TF was transcribed from its own target promoters, each known to hold different input/output transfer functions. In the first case (xylR under the strong promoter of the upper TOL operon, Pu), the reporter system displayed an increased transcriptional capacity in the resulting network for both the optimal and the suboptimal XylR effectors. In contrast, when xylR was expressed under the weaker Ps promoter, the resulting circuit unmistakably discriminated m-xylene from 3MBA. The non-natural connectivity engineered in the network resulted both in a higher promoter activity and also in a much-increased signal-to-background ratio. These results indicate that the working regimes of given genetic circuits can be dramatically altered through simple changes in the way upstream transcription factors are self-regulated by positive or negative feedback loops.

Vyšlo v časopise: Increasing Signal Specificity of the TOL Network of mt-2 by Rewiring the Connectivity of the Master Regulator XylR. PLoS Genet 8(10): e32767. doi:10.1371/journal.pgen.1002963
Kategorie: Research Article
prolekare.web.journal.doi_sk: https://doi.org/10.1371/journal.pgen.1002963

Souhrn

Zdroje

1. PerezJC, GroismanEA (2009) Evolution of transcriptional regulatory circuits in bacteria. Cell 138: 233–244.

2. WallME, HlavacekWS, SavageauMA (2004) Design of gene circuits: lessons from bacteria. Nat Rev Genet 5: 34–42.

3. Silva-RochaR, de LorenzoV (2010) Noise and robustness in prokaryotic regulatory networks. Annu Rev Microbiol 64: 257–275.

4. Van HijumSAFT, MedemaMH, KuipersOP (2009) Mechanisms and evolution of control logic in prokaryotic transcriptional regulation. Microbiol Mol Biol Rev 73: 481–509.

5. Silva-RochaR, TamamesJ, dos SantosVM, de LorenzoV (2011) The logicome of environmental bacteria: merging catabolic and regulatory events with Boolean formalisms. Environ Microbiol 13: 2389–2402.

6. RamosJL, MarquesS (1997) Transcriptional control of the Pseudomonas TOL plasmid catabolic operons is achieved through an interplay of host factors and plasmid-encoded regulators. Annu Rev Microbiol 51: 341–373.

7. Silva-RochaR, de JongH, TamamesJ, de LorenzoV (2011) The logic layout of the TOL network of Pseudomonas putida pWW0 plasmid stems from a metabolic amplifier motif (MAM) that optimizes biodegradation of m-xylene. BMC Syst Biol 5: 191.

8. Silva-RochaR, de LorenzoV (2011) A composite feed-forward loop I4-FFL involving IHF and Crc stabilizes expression of the XylR regulator of Pseudomonas putida mt-2 from growth phase perturbations. Mol Biosyst 7: 2982–2990.

9. InouyeS, NakazawaA, NakazawaT (1987) Expression of the regulatory gene xylS on the TOL plasmid is positively controlled by the xylR gene product. Proc Natl Acad Sci USA 84: 5182–5186.

10. MarquésS, GallegosMT, ManzaneraM, HoltelA, TimmisKN, et al. (1998) Activation and repression of transcription at the double tandem divergent promoters for the xylR and xylS genes of the TOL plasmid of Pseudomonas putida. J Bacteriol 180: 2889–2894.

11. AbrilMA, MichanC, TimmisKN, RamosJL (1989) Regulator and enzyme specificities of the TOL plasmid-encoded upper pathway for degradation of aromatic hydrocarbons and expansion of the substrate range of the pathway. J Bacteriol 171: 6782–6790.

12. Perez-MartinJ, de LorenzoV (1996) VTR expression cassettes for engineering conditional phenotypes in Pseudomonas: activity of the Pu promoter of the TOL plasmid under limiting concentrations of the XylR activator protein. Gene 172: 81–86.

13. FraileS, RoncalF, FernandezLA, de LorenzoV (2001) Monitoring intracellular levels of XylR in Pseudomonas putida with a single-chain antibody specific for aromatic-responsive enhancer-binding proteins. J Bacteriol 183: 5571–5579.

14. JuradoP, FernandezLA, de LorenzoV (2003) Sigma 54 levels and physiological control of the Pseudomonas putida Pu promoter. J Bacteriol 185: 3379–3383.

15. BehzadianF, BarjesteH, HosseinkhaniS, ZareiAR (2011) Construction and characterization of Escherichia coli whole-cell biosensors for toluene and related compounds. Curr Microbiol 62: 690–696.

16. GarmendiaJ, de las HerasA, GalvaoTC, de LorenzoV (2008) Tracing explosives in soil with transcriptional regulators of Pseudomonas putida evolved for responding to nitrotoluenes. Micro Biotech 1: 236–246.

17. de Las HerasA, de LorenzoV (2011) Cooperative amino acid changes shift the response of the sigma-dependent regulator XylR from natural m-xylene towards xenobiotic 2,4-dinitrotoluene. Mol Microbiol 79: 1248–1259.

18. GarmendiaJ, DevosD, ValenciaA, de LorenzoV (2001) A la carte transcriptional regulators: unlocking responses of the prokaryotic enhancer-binding protein XylR to non-natural effectors. Mol Microbiol 42: 47–59.

19. de Las HerasA, de LorenzoV (2012) Engineering whole-cell biosensors with no antibiotic markers for monitoring aromatic compounds in the environment. Methods Mol Biol 834: 261–281.

20. YuQ, LiY, MaA, LiuW, WangH, et al. (2011) An efficient design strategy for a whole-cell biosensor based on engineered ribosome binding sequences. Anal Bioanal Chem 401: 2891–2898.

21. de LorenzoV, HerreroM, MetzkeM, TimmisKN (1991) An upstream XylR- and IHF-induced nucleoprotein complex regulates the sigma 54-dependent Pu promoter of TOL plasmid. EMBO J 10: 1159–1167.

22. RescalliE, SainiS, BartocciC, RychlewskiL, De LorenzoV, et al. (2004) Novel physiological modulation of the Pu promoter of TOL plasmid: negative regulatory role of the TurA protein of Pseudomonas putida in the response to suboptimal growth temperatures. J Biol Chem 279: 7777–7784.

23. DelgadoA, RamosJL (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: 8059–8062.

24. de Las HerasA, ChavarriaM, de LorenzoV (2011) Association of dnt genes of Burkholderia sp. DNT with the substrate-blind regulator DntR draws the evolutionary itinerary of 2,4-dinitrotoluene biodegradation. Mol Microbiol 82: 287–299.

25. LambertsenL, SternbergC, MolinS (2004) Mini-Tn7 transposons for site-specific tagging of bacteria with fluorescent proteins. Environ Microbiol 6: 726–732.

26. GalvãoTC, MencíaM, de LorenzoV (2007) Emergence of novel functions in transcriptional regulators by regression to stem protein types. Mol Microbiol 65: 907–919.

27. O'ShaughnessyEC, PalaniS, CollinsJJ, SarkarCA (2011) Tunable signal processing in synthetic MAP kinase cascades. Cell 144: 119–131.

28. AndersenJB, SternbergC, PoulsenLK, BjornSP, GivskovM, et al. (1998) New unstable variants of green fluorescent protein for studies of transient gene expression in bacteria. Appl Environ Microbiol 64: 2240–2246.

29. AlexcievK, UschevaA, PavlovaM, YavachevL, IvanovI (1989) Expression of synthetic calcitonin genes in plasmid vectors containing tandemly repeated non-overlapping ribosome binding sites. Int J Biochem 21: 987–996.

30. SneppenK, KrishnaS, SemseyS (2010) Simplified models of biological networks. Annu Rev Biophys 39: 43–59.

31. HermsenR, EricksonDW, HwaT (2011) Speed, sensitivity, and bistability in auto-activating signaling circuits. PLoS Comput Biol 7: e1002265 doi:10.1371/journal.pcbi.1002265.

32. ShahNA, SarkarCA (2011) Robust network topologies for generating switch-like cellular responses. PLoS Comput Biol 7: e1002085 doi:10.1371/journal.pcbi.1002085.

33. RaiN, AnandR, RamkumarK, SreenivasanV, DabholkarS, et al. (2012) Prediction by promoter logic in bacterial quorum sensing. PLoS Comput Biol 8: e1002361 doi:10.1371/journal.pcbi.1002361.

34. BertoniG, Perez-MartinJ, de LorenzoV (1997) Genetic evidence of separate repressor and activator activities of the XylR regulator of the TOL plasmid, pWW0, of Pseudomonas putida. Mol Microbiol 23: 1221–1227.

35. MitrophanovAY, GroismanEA (2008) Positive feedback in cellular control systems. Bioessays 30: 542–555.

36. HoltelA, TimmisKN, RamosJL (1992) Upstream binding sequences of the XylR activator protein and integration host factor in the xylS gene promoter region of the Pseudomonas TOL plasmid. Nucl Acids Res 20: 1755–1762.

37. MarquésS, HoltelA, TimmisKN, RamosJL (1994) Transcriptional induction kinetics from the promoters of the catabolic pathways of TOL plasmid pWW0 of Pseudomonas putida for metabolism of aromatics. J Bacteriol 176: 2517–2524.

38. LimWA (2010) Designing customized cell signalling circuits. Nat Rev Mol Cell Biol 11: 393–403.

39. de Las HerasA, CarrenoCA, Martinez-GarciaE, de LorenzoV (2010) Engineering input/output nodes in prokaryotic regulatory circuits. FEMS Microbiol Rev 34: 842–865.

40. WackettLP (2008) Biosensors. Microbial Biotechnology 1: 331–332.

41. van der Meer JR (2011) Bacterial sensors: Synthetic design and application principles; Amos M, editor. New Jersey: Morgan & Claypool.

42. NandagopalN, ElowitzMB (2011) Synthetic Biology: Integrated gene circuits. Science 333: 1244–1248.

43. OlaniranA, MotebejaneR, PillayB (2008) Bacterial biosensors for rapid and effective monitoring of biodegradation of organic pollutants in wastewater effluents. J Environ Monit 10: 889.

44. RonEZ (2007) Biosensing environmental pollution. Curr Opin Biotechnol 18: 252–256.

45. van der MeerJR, BelkinS (2010) Where microbiology meets microengineering: design and applications of reporter bacteria. Nat Rev Microbiol 8: 511–522.

46. LoogerLL, DwyerMA, SmithJJ, HellingaHW (2003) Computational design of receptor and sensor proteins with novel functions. Nature 423: 185–190.

47. DubnauD, LosickR (2006) Bistability in bacteria. Mol Microbiol 61: 564–572.

48. KarigD, WeissR (2005) Signal-amplifying genetic circuit enables in vivo observation of weak promoter activation in the Rhl quorum sensing system. Biotechnol Bioeng 89: 709–718.

49. de Las Heras A, de Lorenzo V (2010) Genetic constructs: Molecular tools for the assembly of environmental bacterial biosensors. In: Timmis KN, editor. Handbook of Hydrocarbon and Lipid Microbiology. Berlin: Springer-Verlag pp. 2651–2676.

50. PedelacqJD, CabantousS, TranT, TerwilligerTC (2006) Engineering and characterization of a superfolder green fluorescent protein. Nature Biotech 24: 79–88.

51. TropelD, van der MeerJR (2004) Bacterial transcriptional regulators for degradation pathways of aromatic compounds. Microbiol Mol Biol Rev 68: 474–500.

52. CamasFM, PoyatosJF (2008) What determines the assembly of transcriptional network motifs in Escherichia coli? PLoS ONE 3: e3657 doi:10.1371/journal.pone.0003657.

53. NelsonKE, WeinelC, PaulsenIT, DodsonRJ, HilbertH, et al. (2002) Complete genome sequence and comparative analysis of the metabolically versatile Pseudomonas putida KT2440. Environ Microbiol 4: 799–808.

54. de LorenzoV, TimmisKN (1994) Analysis and construction of stable phenotypes in gram-negative bacteria with Tn5- and Tn10-derived minitransposons. Methods Enzymol 235: 386–405.

55. Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: A laboratory manual. New York: Cold Spring Harbor.

56. de Las HerasA, CarreñoCA, de LorenzoV (2008) Stable implantation of orthogonal sensor circuits in Gram-negative bacteria for environmental release. Env Microbiol 10: 3305–3316.

57. McKownRL, OrleKA, ChenT, CraigNL (1988) Sequence requirements of Escherichia coli attTn7, a specific site of transposon Tn7 insertion. J Bacteriol 170: 352–358.

58. Sanchez-RomeroJM, Diaz-OrejasR, de LorenzoV (1998) Resistance to tellurite as a selection marker for genetic manipulations of Pseudomonas strains. Appl Env Microbiol 64: 4040–4046.

59. ChoiKH, GaynorJB, WhiteKG, LopezC, BosioCM, et al. (2005) A Tn7-based broad-range bacterial cloning and expression system. Nat Methods 2: 443–448.