DOI QR코드

DOI QR Code

LuxR-Type SCO6993 Negatively Regulates Antibiotic Production at the Transcriptional Stage by Binding to Promoters of Pathway-Specific Regulatory Genes in Streptomyces coelicolor

  • Tsevelkhoroloo, Maral (Department of Bioscience and Bioinformatics, Myongji University) ;
  • Li, Xiaoqiang (Department of Bioscience and Bioinformatics, Myongji University) ;
  • Jin, Xue-Mei (Department of Bioscience and Bioinformatics, Myongji University) ;
  • Shin, Jung-Ho (R&D, Health & Bioscience, DuPont-IFF) ;
  • Lee, Chang-Ro (Department of Bioscience and Bioinformatics, Myongji University) ;
  • Kang, Yup (Institute for Medical Sciences, Ajou University School of Medicine) ;
  • Hong, Soon-Kwang (Department of Bioscience and Bioinformatics, Myongji University)
  • Received : 2022.07.21
  • Accepted : 2022.08.29
  • Published : 2022.09.28

Abstract

SCO6993 (606 amino acids) in Streptomyces coelicolor belongs to the large ATP-binding regulators of the LuxR family regulators having one DNA-binding motif. Our previous findings predicted that SCO6993 may suppress the production of pigmented antibiotics, actinorhodin, and undecylprodigiosin, in S. coelicolor, resulting in the characterization of its properties at the molecular level. SCO6993-disruptant, S. coelicolor ΔSCO6993 produced excess pigments in R2YE plates as early as the third day of culture and showed 9.0-fold and 1.8-fold increased production of actinorhodin and undecylprodigiosin in R2YE broth, respectively, compared with that by the wild strain and S. coelicolor ΔSCO6993/SCO6993+. Real-time polymerase chain reaction analysis showed that the transcription of actA and actII-ORF4 in the actinorhodin biosynthetic gene cluster and that of redD and redQ in the undecylprodigiosin biosynthetic gene cluster were significantly increased by SCO6993-disruptant. Electrophoretic mobility shift assay and DNase footprinting analysis confirmed that SCO6993 protein could bind only to the promoters of pathway-specific transcriptional activator genes, actII-ORF4 and redD, and a specific palindromic sequence is essential for SCO6993 binding. Moreover, SCO6993 bound to two palindromic sequences on its promoter region. These results indicate that SCO6993 suppresses the expression of other biosynthetic genes in the cluster by repressing the transcription of actII-ORF4 and redD and consequently negatively regulating antibiotic production.

Keywords

Acknowledgement

This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT & Future Planning (NRF-2020R1F1A1060789).

References

  1. McCormick JR, Flardh K. 2012. Signals and regulators that govern Streptomyces development. FEMS Microbiol. Rev. 36: 206-231. https://doi.org/10.1111/j.1574-6976.2011.00317.x
  2. Arias P, Fernandez-Moreno MA, Malpartida F. 1999. Characterization of the pathway-specific positive transcriptional regulator for actinorhodin biosynthesis in Streptomyces coelicolor A3(2) as a DNA-binding protein. J. Bacteriol.181: 6958-6968. https://doi.org/10.1128/jb.181.22.6958-6968.1999
  3. Uguru GC, Stephens KE, Stead JA, Towle JE, Baumberg S, McDowall KJ. 2005. Transcriptional activation of the pathway-specific regulator of the actinorhodin biosynthetic genes in Streptomyces coelicolor. Mol. Microbiol. 58: 131-150. https://doi.org/10.1111/j.1365-2958.2005.04817.x
  4. Ryding NJ, Anderson TB, Champness WC. 2002. Regulation of the Streptomyces coelicolor calcium-dependent antibiotic by absA, encoding a cluster-linked two-component system. J. Bacteriol. 184: 794-805. https://doi.org/10.1128/JB.184.3.794-805.2002
  5. Hong SK, Kito M, Beppu T, Horinouchi S. 1991. Phosphorylation of the AfsR product, a global regulatory protein for secondary-metabolite formation in Streptomyces coelicolor A3(2). J. Bacteriol. 173: 2311-2318. https://doi.org/10.1128/jb.173.7.2311-2318.1991
  6. Chater KF. 2013. Curing baldness activates antibiotic production. Chem. Biol. 20: 1199-1200. https://doi.org/10.1016/j.chembiol.2013.10.001
  7. Horinouchi S, Hara O, Beppu T.1983. Cloning of a pleiotropic gene that positively controls biosynthesis of A-factor, actinorhodin, and prodigiosin in Streptomyces coelicolor A3(2) and Streptomyces lividans. J. Bacteriol. 155: 1238-1248. https://doi.org/10.1128/jb.155.3.1238-1248.1983
  8. Horinouchi S. 2003. AfsR as an integrator of signals that are sensed by multiple serine/threonine kinases in Streptomyces coelicolor A3(2). J. Ind. Microbiol. Biotechnol. 30: 462-467. https://doi.org/10.1007/s10295-003-0063-z
  9. White J, Bibb M. 1997. bldA dependence of undecylprodigiosin production in Streptomyces coelicolor A3(2) involves a pathway-specific regulatory cascade. J. Bacteriol.179: 627-633. https://doi.org/10.1128/jb.179.3.627-633.1997
  10. McKenzie NL, Nodwell JR. 2007. Phosphorylated AbsA2 negatively regulates antibiotic production in Streptomyces coelicolor through interactions with pathway-specific regulatory gene promoters. J. Bacteriol. 189: 5284-5292. https://doi.org/10.1128/JB.00305-07
  11. van der Heul HU, Bilyk BL, McDowall KJ, Seipke RF, van Wezel GP. 2018. Regulation of antibiotic production in actinobacteria: new perspectives from the post-genomic era. Nat. Prod. Rep. 35: 575-604. https://doi.org/10.1039/c8np00012c
  12. Jin XM, Choi MY, Tsevelkhoroloo M, Park U, Suh JW, Hong SK. 2021. SCO6992, a protein with β-glucuronidase activity, complements a mutation at the absR locus and promotes antibiotic biosynthesis in Streptomyces coelicolor. J. Microbiol. Biotechnol. 31: 1591-1600. https://doi.org/10.4014/jmb.2108.08001
  13. Anderson TB, Brian P, Champness WC. 2001. Genetic and transcriptional analysis of absA, an antibiotic gene cluster-linked two-component system that regulates multiple antibiotics in Streptomyces coelicolor. Mol. Microbiol. 39: 553-566. https://doi.org/10.1046/j.1365-2958.2001.02240.x
  14. Mazodier P, Petter R, Thompson C. 1989. Intergeneric conjugation between Escherichia coli and Streptomyces species. J. Bacteriol. 71: 3583-3585. https://doi.org/10.1128/jb.171.6.3583-3585.1989
  15. Datsenko KA, Wanner BL. 2000. One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc. Natl. Acad. Sci. USA 97: 6640-6645. https://doi.org/10.1073/pnas.120163297
  16. Sambrook J, and Russell DW. 2001. Molecular cloning: a laboratory manual. 3rd Edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
  17. Kieser H, Bibb MJ, Buttner MJ, Chater FK, Hopwood DA. 2000. Practical Streptomyces Genetics. The John Innes Foundation, Norwich, UK.
  18. Gust B, Challis GL, Fowler K, Kieser T, Chater KF. 2003. PCR-targeted Streptomyces gene replacement identifies a protein domain needed for biosynthesis of the sesquiterpene soil odor geosmin. Proc. Natl. Acad. Sci. USA 100: 1541-1546. https://doi.org/10.1073/pnas.0337542100
  19. Yindeeyoungyeon W, Schell MA. 2000. Footprinting with an automated capillary DNA sequencer. Biotechniques 29: 1034-1036. https://doi.org/10.2144/00295st05
  20. Newman JD, Russell MM, Fan L, Wang YX, Gonzalez-Gutierrez G, van Kessel JC. 2021. The DNA binding domain of the Vibrio vulnificus SmcR transcription factor is flexible and binds diverse DNA sequences. Nucleic Acids Res. 49: 5967-5984. https://doi.org/10.1093/nar/gkab387
  21. Engebrecht J, Nealson K, Silverman M. 1983. Bacterial bioluminescence: isolation and genetic analysis of functions from Vibrio fischeri. Cell 32: 773-781. https://doi.org/10.1016/0092-8674(83)90063-6
  22. Fuqua C, Greenberg EP. 2002. Listening in on bacteria: acyl-homoserine lactone signalling. Nat. Rev. Mol. Cell. Biol. 3: 685-695. https://doi.org/10.1038/nrm907
  23. Fuqua C, Winans SC, Greenberg EP. 1996. Census and consensus in bacterial ecosystems: the LuxR-LuxI family of quorum-sensing transcriptional regulators. Annu. Rev. Microbiol. 50: 727-751. https://doi.org/10.1146/annurev.micro.50.1.727
  24. Patankar AV, Gonzalez JE. 2009. Orphan LuxR regulators of quorum sensing. FEMS Microbiol. Rev. 33: 739-756. https://doi.org/10.1111/j.1574-6976.2009.00163.x
  25. Boos W, Shuman H. 1998. Maltose/maltodextrin system of Escherichia coli: transport, metabolism, and regulation. Microbiol. Mol. Biol. Rev. 62: 204-229. https://doi.org/10.1128/mmbr.62.1.204-229.1998
  26. Walker JE, Saraste M, Runswick MJ, Gay NJ. 1982. Distantly related sequences in the alpha- and beta-subunits of ATP synthase, myosin, kinases and other ATP-requiring enzymes and a common nucleotide binding fold. EMBO J. 1: 945-951. https://doi.org/10.1002/j.1460-2075.1982.tb01276.x
  27. Raibaud O, Vidal-Ingigliardi D, Richet EA. 1989. Complex nucleoprotein structure involved in activation of transcription of two divergent Escherichia coli promoters. J. Mol. Biol. 205: 471-485. https://doi.org/10.1016/0022-2836(89)90218-0
  28. Maris AE, Sawaya MR, Kaczor-Grzeskowiak M, Jarvis MR, Bearson SM, Kopka ML, et al. 2022. Dimerization allows DNA target site recognition by the NarL response regulator. Nat. Struct. Biol. 9: 771-778. https://doi.org/10.1038/nsb845
  29. Wilson DJ, Xue Y, Reynolds KE, Sherman DH. 2001. Characterization and analysis of the PikD regulatory factor in the pikromycin biosynthetic pathway of Streptomyces venezuelae. J. Bacteriol.183: 3468-3475. https://doi.org/10.1128/JB.183.11.3468-3475.2001
  30. Aparicio JF, Molnar I, Schwecke T, Konig A, Haydock SF, Khaw LE, et al. 1996. Organisation of the biosynthetic gene cluster for rapamycin in Streptomyces hygroscopicus: analysis of the enzymatic domains in the modular polyketide synthase. Gene 169: 9-16. https://doi.org/10.1016/0378-1119(95)00800-4
  31. Sekurova ON, Brautaset T, Sletta H, Borgos SEF, Jakobsen OM, Ellingsen TE, et al. 2004. In vivo analysis of the regulatory genes in the nystatin biosynthetic gene cluster of Streptomyces noursei ATCC 11455 reveals their differential control over antibiotic biosynthesis. J. Bacteriol. 186: 1345-1354. https://doi.org/10.1128/JB.186.5.1345-1354.2004
  32. Hur YA, Choi SS, Sherman DH, Kim ES. 2008. Identification of TmcN as a pathway-specific positive regulator of tautomycetin biosynthesis in Streptomyces sp. CK4412. Microbiology 54: 2912-2919.
  33. He X, Li R, Pan Y, Liu G, Tan H. 2010. SanG, a transcriptional activator, controls nikkomycin biosynthesis through binding to the sanN-sanO intergenic region in Streptomyces ansochromogenes. Microbiology 156: 828-837. https://doi.org/10.1099/mic.0.033605-0
  34. Mo X, Wang Z, Wang B, Ma J, Huang H, Tian X, Zhang S, et al. 2011. Cloning and characterization of the biosynthetic gene cluster of the bacterial RNA polymerase inhibitor tirandamycin from marine-derived Streptomyces sp. SCSIO1666. Biochem. Biophys. Res. Commun. 406: 341-347. https://doi.org/10.1016/j.bbrc.2011.02.040
  35. Carmody M, Byrne B, Murphy B, Breen C, Lynch S, Flood E, et al. 2004. Analysis and manipulation of amphotericin biosynthetic genes by means of modified phage KC515 transduction techniques. Gene 343: 107-115. https://doi.org/10.1016/j.gene.2004.08.006
  36. Santos-Aberturas J, Vicente CM, Payero TD, Martin-Sanchez L, Canibano C, Martin JF, et al. 2012. Hierarchical control on polyene macrolide biosynthesis: PimR modulates pimaricin production via the PAS-LuxR transcriptional activator PimM. PLoS One 7: e38536. https://doi.org/10.1371/journal.pone.0038536
  37. Guerra SM, Rodriguez-Garcia A, Santos-Aberturas J, Vicente CM, Payero TD, Martin JF, et al. 2012. LAL regulators SCO0877 and SCO7173 as pleiotropic modulators of phosphate starvation response and actinorhodin biosynthesis in Streptomyces coelicolor. PLoS One 7: e31475. https://doi.org/10.1371/journal.pone.0031475
  38. Guo J, Zhao J, Li L, Chen Z, Wen Y, Li J. 2010.The pathway-specific regulator AveR from Streptomyces avermitilis positively regulates avermectin production while it negatively affects oligomycin biosynthesis. Mol. Genet. Genomics 283: 123-133. https://doi.org/10.1007/s00438-009-0502-2
  39. Hackl S, Bechthold A. 2015. The Gene bldA, a regulator of morphological differentiation and antibiotic production in Streptomyces. Arch. Pharm. (Weinheim) 348: 455-462. https://doi.org/10.1002/ardp.201500073