Advertisement

Applied Microbiology and Biotechnology

, Volume 104, Issue 3, pp 1163–1174 | Cite as

A plasmid-based genomic screening system for transcriptional regulators of non-adjacent xenobiotic catabolism genes

  • Minggen Cheng
  • Ziyu Xing
  • Luyao Lu
  • Feng Chen
  • Jian He
  • Xing HuangEmail author
Applied genetics and molecular biotechnology
  • 98 Downloads

Abstract

Bacteria play an important role in the catabolism of environmental xenobiotics. The study of transcriptional regulation has greatly enhanced our understanding of the molecular mechanisms associated with these processes. However, genes encoding transcription factors (TFs) for xenobiotic catabolism are usually not located in the immediate vicinity of the catabolic genes that they regulate; therefore, functional identification of these TFs is difficult. Significantly modified from a metagenome screening method substrate-induced gene expression (SIGEX), here we propose a synthetic pSRGFP-18 plasmid-based tool as a TF reporter system. In short, two multiple cloning sites (MCS) were designed; one in front of an egfp reporter gene was constructed for promoter insertion, and the other MCS was used for shotgun cloning of genomic DNA. Based on the regulatory relationship between TFs and the promoter of their associated catabolic genes, this approach allowed us to screen for TF genes using a genome shotgun approach. This system performed well when testing the known operons. Following statistical analysis of known catabolic operons in Escherichia coli and Bacillus subtilis, the suggested region of the target promoter for this system was from − 250 to + 150. Furthermore, to broaden the applicability of this plasmid, a series of pSRGFP-18 and pBBR1-based pSRGFP-X plasmids were constructed, which had broad host ranges and contained different antibiotic markers. This study outlines a powerful tool to enable functional identification of TFs for bacterial xenobiotic catabolism.

Keywords

Transcriptional regulatory genes Xenobiotic catabolism Shotgun screening pSRGFP-18 pSRGFP-X 

Notes

Funding information

This work was funded by the National Natural Science Foundation of China (41671317, 41977119) and the Fundamental Research Funds for the Central Universities (KJQN201940).

Compliance with ethical standards

Ethics statement

This article does not contain any studies with human participants or animals performed by any of the authors.

Conflict of interest

The authors declare that they have no conflict of interest.

References

  1. Akers JC, HoDac H, Lathrop RH, Tan M (2011) Identification and functional analysis of CT069 as a novel transcriptional regulator in Chlamydia. J Bacteriol 193(22):6123–6131.  https://doi.org/10.1128/JB.05976-11 CrossRefPubMedPubMedCentralGoogle Scholar
  2. Babu MM (2013) Bacterial gene regulation and transcriptional networks. Caister Academic Press, MRC Laboratory of Molecular Biology, CambridgeGoogle Scholar
  3. Bagdasarian M, Lurz R, Rückert B, Franklin FCH, Bagdasarian MM, Frey J, Timmis KN (1981) Specific-purpose plasmid cloning vectors ii. Broad host range, high copy number, RSF 1010-derived vectors, and a host-vector system for gene cloning in Pseudomonas. Gene 16(1–3):237–247.  https://doi.org/10.1016/0378-1119(81)90080-9 CrossRefPubMedGoogle Scholar
  4. Bethesda Research Laboratories (1986) BRL pUC host: E. coli DH5α competent cells. Bethesda Res Lab Focus 8(2):9Google Scholar
  5. Chao H, Zhou N (2013) GenR, an IclR-type regulator, activates and represses the transcription of gen genes involved in 3-hydroxybenzoate and gentisate catabolism in Corynebacterium glutamicum. J Bacteriol 195(7):1598–1609.  https://doi.org/10.1128/JB.02216-12 CrossRefPubMedPubMedCentralGoogle Scholar
  6. Chen YJ, Liu P, Nielsen AA, Brophy JA, Clancy K, Peterson T, Voigt CA (2013) Characterization of 582 natural and synthetic terminators and quantification of their design constraints. Nat Methods 10(7):659–664.  https://doi.org/10.1038/nmeth.2515 CrossRefPubMedGoogle Scholar
  7. Cheng M, Chen K, Guo S, Huang X, He J, Li S, Jiang J (2015) PbaR, an IclR family transcriptional activator for the regulation of the 3-phenoxybenzoate 1′,2′-dioxygenase gene cluster in Sphingobium wenxiniae JZ-1T. Appl Environ Microbiol 81(23):8084–8092.  https://doi.org/10.1128/AEM.02122-15 CrossRefPubMedPubMedCentralGoogle Scholar
  8. Cheng M, Meng Q, Yang Y, Chu C, Chen Q, Li Y, Cheng D, Hong Q, Yan X, He J (2017) The two-component monooxygenase MeaXY initiates the downstream pathway of chloroacetanilide herbicide catabolism in sphingomonads. Appl Environ Microbiol 83(7):AEM.03241-16.  https://doi.org/10.1128/AEM.03241-16 CrossRefGoogle Scholar
  9. Cowles CE, Nichols NN, Harwood CS (2000) BenR, a XylS homologue, regulates three different pathways of aromatic acid degradation in Pseudomonas putida. J Bacteriol 182(22):6339–6346.  https://doi.org/10.1128/jb.182.22.6339-6346.2000 CrossRefPubMedPubMedCentralGoogle Scholar
  10. Cramer A, Gerstmeir R, Schaffer S, Bott M, Eikmanns BJ (2006) Identification of RamA, a novel LuxR-type transcriptional regulator of genes involved in acetate metabolism of Corynebacterium glutamicum. J Bacteriol 188(7):2554–2567.  https://doi.org/10.1128/JB.188.7.2554-2567.2006 CrossRefPubMedPubMedCentralGoogle Scholar
  11. Díaz E (2008) Microbial biodegradation: genomics and molecular biology. Caister Academic Press, NorfolkGoogle Scholar
  12. Gertz J, Siggia ED, Cohen BA (2009) Analysis of combinatorial cis-regulation in synthetic and genomic promoters. Nature 457(7226):215–218.  https://doi.org/10.1038/nature07521 CrossRefPubMedGoogle Scholar
  13. Horbal L, Fedorenko V, Bechthold A, Luzhetskyy A (2013) A transposon-based strategy to identify the regulatory gene network responsible for landomycin E biosynthesis. FEMS Microbiol Lett 342(2):138–146.  https://doi.org/10.1111/1574-6968.12117 CrossRefPubMedGoogle Scholar
  14. Hyeon JE, Kang DH, Kim YI, You SK, Han SO (2012) GntR-type transcriptional regulator PckR negatively regulates the expression of phosphoenolpyruvate carboxykinase in Corynebacterium glutamicum. J Bacteriol 194(9):2181–2188.  https://doi.org/10.1128/JB.06562-11 CrossRefPubMedPubMedCentralGoogle Scholar
  15. Jiménez JI, Canales A, Jiménez-Barbero J, Ginalski K, Rychlewski L, García JL, Díaz E (2008) Deciphering the genetic determinants for aerobic nicotinic acid degradation: the nic cluster from Pseudomonas putida KT2440. Proc Natl Acad Sci U S A 105(32):11329–11334.  https://doi.org/10.1073/pnas.0802273105 CrossRefPubMedPubMedCentralGoogle Scholar
  16. Jiménez JI, Juárez JF, García JL, Díaz E (2011) A finely tuned regulatory circuit of the nicotinic acid degradation pathway in Pseudomonas putida. Environ Microbiol 13(7):1718–1732.  https://doi.org/10.1111/j.1462-2920.2011.02471.x CrossRefPubMedGoogle Scholar
  17. Kadonaga JT, Tjian R (1986) Affinity purification of sequence-specific DNA binding proteins. Proc Natl Acad Sci U S A 83(16):5889–5893.  https://doi.org/10.1073/pnas.83.16.5889 CrossRefPubMedPubMedCentralGoogle Scholar
  18. Kamimura N, Inakazu K, Kasai D, Fukuda M, Masai E (2012) Regulation of the isophthalate catabolic operon controlled by IphR in Comamonas sp. strain E6. FEMS Microbiol Lett 329(2):186–192.  https://doi.org/10.1111/j.1574-6968.2012.02521.x CrossRefPubMedGoogle Scholar
  19. Kovach ME, Elzer PH, Hill DS, Robertson GT, Farris MA, Roop RM, Peterson KM (1995) Four new derivatives of the broad-host-range cloning vector pBBR1MCS, carrying different antibiotic-resistance cassettes. Gene 166(1):175–176.  https://doi.org/10.1016/0378-1119(95)00584-1 CrossRefPubMedPubMedCentralGoogle Scholar
  20. Norrander J, Kempe T, Messing J (1983) Construction of improved M13 vectors using oligodeoxynucleotide-directed mutagenesis. Gene 26(1):101–106.  https://doi.org/10.1016/0378-1119(83)90040-9 CrossRefPubMedGoogle Scholar
  21. Russell JB, Cook GM (1995) Energetics of bacterial growth: balance of anabolic and catabolic reactions. Microbiol Rev 59(1):48–62PubMedPubMedCentralGoogle Scholar
  22. Salgado H, Peralta-Gil M, Gama-Castro S, Santos-Zavaleta A, Muñiz-Rascado L, García-Sotelo JS, Weiss V, Solano-Lira H, Martínez-Flores I, Medina-Rivera A, Salgado-Osorio G, Alquicira-Hernández S, Alquicira-Hernández K, López-Fuentes A, Porrón-Sotelo L, Huerta AM, Bonavides-Martínez C, Balderas-Martínez YI, Pannier L, Olvera M, Labastida A, Jiménez-Jacinto V, Vega-Alvarado L, del Moral-Chávez V, Hernández-Alvarez A, Morett E, Collado-Vides J (2013) RegulonDB v8.0: omics data sets, evolutionary conservation, regulatory phrases, cross-validated gold standards and more. Nucleic Acids Res 41(Database issue):D203–D213.  https://doi.org/10.1093/nar/gks1201 CrossRefPubMedGoogle Scholar
  23. Sierro N, Makita Y, de Hoon M, Nakai K (2008) DBTBS: a database of transcriptional regulation in Bacillus subtilis containing upstream intergenic conservation information. Nucleic Acids Res 36(Database issue):D93–D96.  https://doi.org/10.1093/nar/gkm910 CrossRefPubMedGoogle Scholar
  24. Tompa M, Li N, Bailey TL, Church GM, De Moor B, Eskin E, Favorov AV, Frith MC, Fu Y, Kent WJ, Makeev VJ, Mironov AA, Noble WS, Pavesi G, Pesole G, Régnier M, Simonis N, Sinha S, Thijs G, van Helden J, Vandenbogaert M, Weng Z, Workman C, Ye C, Zhu Z (2005) Assessing computational tools for the discovery of transcription factor binding sites. Nat Biotechnol 23(1):137–144.  https://doi.org/10.1038/nbt1053 CrossRefPubMedGoogle Scholar
  25. Uchiyama T, Watanabe K (2008) Substrate-induced gene expression (SIGEX) screening of metagenome libraries. Nat Protoc 3(7):1202–1212.  https://doi.org/10.1038/nprot.2008.96 CrossRefPubMedGoogle Scholar
  26. van Hijum SA, Medema MH, Kuipers OP (2009) Mechanisms and evolution of control logic in prokaryotic transcriptional regulation. Microbiol Mol Biol Rev 73(3):481–509.  https://doi.org/10.1128/MMBR.00037-08 CrossRefPubMedPubMedCentralGoogle Scholar
  27. Wang G, Li R, Li S, Jiang J (2010) A novel hydrolytic dehalogenase for the chlorinated aromatic compound chlorothalonil. J Bacteriol 192(11):2737–2745.  https://doi.org/10.1128/JB.01547-09 CrossRefPubMedPubMedCentralGoogle Scholar
  28. Wargo MJ, Szwergold BS, Hogan DA (2008) Identification of two gene clusters and a transcriptional regulator required for Pseudomonas aeruginosa glycine betaine catabolism. J Bacteriol 190(8):2690–2699.  https://doi.org/10.1128/JB.01393-07 CrossRefPubMedGoogle Scholar
  29. Zhang WM, Zhang JJ, Jiang X, Chao H, Zhou NY (2014) Transcriptional activation of multiple operons involved in para-nitrophenol degradation by Pseudomonas sp. strain WBC-3. Appl Environ Microbiol 81(1):220–230.  https://doi.org/10.1128/AEM.02720-14 CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  1. 1.College of Life SciencesNanjing Agricultural UniversityNanjingChina

Personalised recommendations