Advertisement

Negative regulation of bleomycins biosynthesis by ArsR/SmtB family repressor BlmR in Streptomyces verticillus

  • Hong Chen
  • Junhua Wang
  • Jiaqi Cui
  • Cheng Wang
  • Shaoxiong Liang
  • Huanhuan Liu
  • Jianping WenEmail author
Applied genetics and molecular biotechnology

Abstract

Bleomycin, a broad-spectrum antibiotic, has been widely used for various tumor treatments. However, its poor fermentation yield is not satisfactory for industrial production. Here, the ArsR/SmtB family regulator BlmR was characterized as a repressor of bleomycin production. As an autoregulator, BlmR was found to bind to a 12-2-12 imperfect palindrome sequence in its own promoter, and deletion of blmR led to a 34% increase of bleomycin B2 production compared with the wild-type strain. Using reverse transcription and quantitative PCR (RT-qPCR), blmT, which encoded a putative transporter, was identified as the target gene regulated by BlmR. Therefore, high-production strain was constructed by blmT overexpression in a blmR deletion strain, and the bleomycin B2 titer reached to 80 mg/L, which was 1.9-fold higher than the wild-type strain. Moreover, electrophoretic mobility shift assay (EMSA) showed neither metal-binding motifs nor redox switches in BlmR. In order to elucidate the regulatory mechanism, a model of BlmR was constructed by homology modeling and protein-protein docking. The BlmR-DNA complex was generated by protein-DNA docking with the assistance of site-directed mutagenesis and molecular dynamic (MD) simulation, which directly revealed several key amino acid residues needed for the maintenance and stabilization of the interface between BlmR and target DNA. The interface information could provide the configuration reference and seek the potential effectors that could interact with BlmR, thereby extending the regulation role of ArsR/SmtB family members on the improvement of antibiotic production.

Keywords

Bleomycins ArsR/SmtB family regulator Molecular dynamic simulation Key amino acid residues 

Notes

Funding information

This work was financially supported by the National Natural Science Foundation of China (No. 21676189) and the Key Technologies R&D Program of Tianjin (No. 16YFZCSY00780).

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval

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

Supplementary material

253_2019_9923_MOESM1_ESM.pdf (708 kb)
ESM 1 (PDF 707 kb)

References

  1. Alper SL, Sharma AK (2013) The SLC26 gene family of anion transporters and channels. Mol Asp Med 34(2-3):494–515.  https://doi.org/10.1016/j.mam.2012.07.009 CrossRefGoogle Scholar
  2. Aravind L, Anantharaman V, Balaji S, Babu MM, Iyer LM (2010) The many faces of the helix-turn-helix domain: transcription regulation and beyond. FEMS Microbiol Rev 29(2):231–262.  https://doi.org/10.1016/j.fmrre.2004.12.008 CrossRefGoogle Scholar
  3. Arunkumar AI, Campanello GC, Giedroc DP (2009) Solution structure of a paradigm ArsR family zinc sensor in the DNA-bound state. Proc Natl Acad Sci U S A 106(43):18177–18182.  https://doi.org/10.1073/pnas.0905558106 CrossRefGoogle Scholar
  4. Babu M, Greenblatt JF, Emili A, Strynadka NC, Reithmeier RA, Moraes TF (2010) Structure of a SLC26 anion transporter STAS domain in complex with acyl carrier protein: implications for E. coli YchM in fatty acid metabolism. Structure 18(11):1450–1462.  https://doi.org/10.1016/j.str.2010.08.015 CrossRefGoogle Scholar
  5. Bose M, Slick D, Sarto MJ, Murphy P, Roberts D, Roberts J, Barber RD (2014) Identification of SmtB/ArsR cis elements and proteins in archaea using the Prokaryotic InterGenic Exploration Database (PIGED). Archaea 2(1):39–49.  https://doi.org/10.1155/2006/837139 CrossRefGoogle Scholar
  6. Burger RM, Peisach J, Horwitz SB (1981) Activated bleomycin. A transient complex of drug, iron, and oxygen that degrades DNA. J Biol Chem 256(22):11636–11644.  https://doi.org/10.1016/0165-022X(81)90075-0 Google Scholar
  7. Busenlehner LS, Pennella MA, Giedroc DP (2010) The SmtB/ArsR family of metalloregulatory transcriptional repressors: structural insights into prokaryotic metal resistance. FEMS Microbiol Rev 27(2-3):131–143.  https://doi.org/10.1016/S0168-6445(03)00054-8 CrossRefGoogle Scholar
  8. Chauhan S, Kumar A, Singhal A, Tyagi JS, Prasad HK (2010) CmtR, a cadmium-sensing ArsR–SmtB repressor, cooperatively interacts with multiple operator sites to autorepress its transcription in Mycobacterium tuberculosis. FEBS J 276(13):3428–3439.  https://doi.org/10.1111/j.1742-4658.2009.07066.x CrossRefGoogle Scholar
  9. Contursi P, Farina B, Pirone L, Fusco S, Russo L, Bartolucci S, Fattorusso R, Pedone E (2014) Structural and functional studies of Stf76 from the Sulfolobus islandicus plasmid-virus pSSVx: a novel peculiar member of the winged helix-turn-helix transcription factor family. Nucleic Acids Res 42(9):5993–6011.  https://doi.org/10.1093/nar/gku215 CrossRefGoogle Scholar
  10. Coulocheri SA, Pigis DG, Papavassiliou KA, Papavassiliou AG (2007) Hydrogen bonds in protein-DNA complexes: where geometry meets plasticity. Biochimie 89(11):1291–1303.  https://doi.org/10.1016/j.biochi.2007.07.020 CrossRefGoogle Scholar
  11. Dijk M, Bonvin AMJJ (2009) 3D-DART: a DNA structure modelling server. Nucleic Acids Res 37(Web Server issue):W235–W239.  https://doi.org/10.1093/nar/gkp287
  12. Du L, Sánchez C, Chen M, Edwards DJ, Shen B (2000) The biosynthetic gene cluster for the antitumor drug bleomycin from Streptomyces verticillus ATCC15003 supporting functional interactions between nonribosomal peptide synthetases and a polyketide synthase. Chem Biol 7(8):623–642.  https://doi.org/10.1016/S1074-5521(00)00011-9 CrossRefGoogle Scholar
  13. Eicken C, Pennella MA, Chen X, Koshlap KM, Vanzile ML, Sacchettini JC, Giedroc DP (2003) A metal–ligand-mediated intersubunit allosteric switch in related SmtB/ArsR zinc sensor proteins. J Mol Biol 333(4):683–695.  https://doi.org/10.1016/j.jmb.2003.09.007 CrossRefGoogle Scholar
  14. Fineran PC, Everson L, Slater H, Salmond GP (2005) A GntR family transcriptional regulator (PigT) controls gluconate-mediated repression and defines a new, independent pathway for regulation of the tripyrrole antibiotic, prodigiosin, in Serratia. Microbiology 151(Pt 12:3833–3845.  https://doi.org/10.1099/mic.0.28251-0 CrossRefGoogle Scholar
  15. Galagan JE, Minch K, Peterson M, Lyubetskaya A, Azizi E, Sweet L, Gomes A, Rustad T, Dolganov G, Glotova I (2013) The Mycobacterium tuberculosis regulatory network and hypoxia. Nature 499(7457):178–183.  https://doi.org/10.1038/nature12337 CrossRefGoogle Scholar
  16. Galm U, Hager MH, Van Lanen SG, Ju J, Thorson JS, Shen B (2005) Antitumor antibiotics: bleomycin, enediynes, and mitomycin. Chem Rev 105(2):739–758.  https://doi.org/10.1021/cr030117g CrossRefGoogle Scholar
  17. Galm U, Wang L, Wendt-Pienkowski E, Yang R, Liu W, Tao M, Coughlin JM, Shen B (2008) In vivo manipulation of the bleomycin biosynthetic gene cluster in Streptomyces verticillus ATCC15003 revealing new insights into its biosynthetic pathway. J Biol Chem 283(42):28236–28245.  https://doi.org/10.1074/jbc.M804971200 CrossRefGoogle Scholar
  18. Galm U, Wendtpienkowski E, Wang L, Huang SX, Unsin C, Tao M, Coughlin JM, Shen B (2011) Comparative analysis of the biosynthetic gene clusters and pathways for three structurally related antitumor antibiotics: bleomycin, tallysomycin, and zorbamycin. J Nat Prod 74(3):526–536.  https://doi.org/10.1021/np1008152 CrossRefGoogle Scholar
  19. Guimarães BG, Barbosa RL, Soprano AS, Campos BM, De TS, Tonoli CC, Leme AF, Murakami MT, Benedetti CE (2011) Plant pathogenic bacteria utilize biofilm growth-associated repressor (BigR), a novel winged-helix redox switch, to control hydrogen sulfide detoxification under hypoxia. J Biol Chem 286(29):26148–26157.  https://doi.org/10.1074/jbc.M111.234039 CrossRefGoogle Scholar
  20. 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 U S A 100(4):1541–1546.  https://doi.org/10.1073/pnas.0337542100 CrossRefGoogle Scholar
  21. He H, Bretl DJ, Penoske RM, Anderson DM, Zahrt TC (2011) Components of the Rv0081-Rv0088 locus, which encodes a predicted formate hydrogenlyase complex, are coregulated by Rv0081, MprA, and DosR in Mycobacterium tuberculosis. J Bacteriol 193(19):5105–5118.  https://doi.org/10.1128/JB.05562-11 CrossRefGoogle Scholar
  22. Kim HM, Ahn BE, Lee JH, Roe JH (2015) Regulation of a nickel-cobalt efflux system and nickel homeostasis in a soil actinobacterium Streptomyces coelicolor. Metallomics 7(4):702–709.  https://doi.org/10.1039/c4mt00318g CrossRefGoogle Scholar
  23. Lee SG, Krishnan HB, Jez JM (2014) Structural basis for regulation of rhizobial nodulation and symbiosis gene expression by the regulatory protein NolR. Proc Natl Acad Sci U S A 111(17):6509–6514.  https://doi.org/10.1073/pnas.1402243111 CrossRefGoogle Scholar
  24. Liu M, Naka H, Crosa JH (2010) HlyU acts as an H-NS antirepressor in the regulation of the RTX toxin gene essential for the virulence of the human pathogen Vibrio vulnificus CMCP6. Mol Microbiol 72(2):491–505.  https://doi.org/10.1111/j.1365-2958.2009.06664.x CrossRefGoogle Scholar
  25. Lougher MJ (2015) Functional and structural insights into MmyJ, An ArsR-like transcriptional repressor. Dissertation, University of WarwickGoogle Scholar
  26. Ma D, Wang C, Chen H, Wen J (2018) Manipulating the expression of SARP family regulator BulZ and its target gene product to increase tacrolimus production. Appl Microbiol Biotechnol 102(11):4887–4900.  https://doi.org/10.1007/s00253-018-8979-4 CrossRefGoogle Scholar
  27. Mandelgutfreund Y, Schueler O, Margalit H (1995) Comprehensive analysis of hydrogen bonds in regulatory protein DNA-complexes: in search of common principles. J Mol Biol 253(2):370–382.  https://doi.org/10.1006/jmbi.1995.0559 CrossRefGoogle Scholar
  28. Mao XM, Luo S, Li YQ (2017) Negative regulation of daptomycin production by DepR2, an ArsR-family transcriptional factor. J Ind Microbiol Biotechnol 44(12):1653–1658.  https://doi.org/10.1007/s10295-017-1983-3 CrossRefGoogle Scholar
  29. Mou X, Spinard EJ, Driscoll MV, Zhao W, Nelson DR (2013) H-NS is a negative regulator of the two hemolysin/cytotoxin gene clusters in Vibrio anguillarum. Infect Immun 81(10):3566–3576.  https://doi.org/10.1128/IAI.00506-13 CrossRefGoogle Scholar
  30. Mukherjee D, Datta AB, Chakrabarti P (2014) Crystal structure of HlyU, the hemolysin gene transcription activator, from Vibrio cholerae N16961 and functional implications. Biochim Biophys Acta 1844(12):2346–2354.  https://doi.org/10.1016/j.bbaapa.2014.09.020
  31. Mukherjee D, Pal A, Chakravarty D, Chakrabarti P (2015) Identification of the target DNA sequence and characterization of DNA binding features of HlyU, and suggestion of a redox switch for hlyA expression in the human pathogen Vibrio cholerae from in silico studies. Nucleic Acids Res 43(3):1407–1417.  https://doi.org/10.1093/nar/gku1319 CrossRefGoogle Scholar
  32. Murray V, Chen J, Long C (2018) The interaction of the metallo-glycopeptide anti-tumour drug bleomycin with DNA. Int J Mol Sci 19(5):1372.  https://doi.org/10.3390/ijms19051372 CrossRefGoogle Scholar
  33. Osman D, Cavet JS (2010) Bacterial metal-sensing proteins exemplified by ArsR-SmtB family repressors. Nat Prod Rep 27(5):668–680.  https://doi.org/10.1039/b906682a CrossRefGoogle Scholar
  34. Palmano S, Firrao G, Locci R (2000) Sequence analysis of domains III and IV of the 23S rRNA gene of verticillate streptomycetes. Int J Syst Evol Microbiol 50(3):1187–1191.  https://doi.org/10.1099/00207713-50-3-1187 CrossRefGoogle Scholar
  35. Rohs R, Jin X, West SM, Joshi R, Honig B, Mann RS (2010) Origins of specificity in protein-DNA recognition. Annu Rev Biochem 79(1):233–269.  https://doi.org/10.1146/annurev-biochem-060408-091030 CrossRefGoogle Scholar
  36. Roy R, Samanta S, Patra S, Mahato NK, Saha RP (2018) In silico identification and characterization of sensory motifs in the transcriptional regulators of the ArsR-SmtB family. Metallomics 10:1476–1500.  https://doi.org/10.1039/C8MT00082D CrossRefGoogle Scholar
  37. Shi W, Dong J, Scott RA, Ksenzenko MY, Rosen BP (1996) The role of arsenic-thiol interactions in metalloregulation of the ars Operon. J Biol Chem 271(16):9291–9297.  https://doi.org/10.1074/jbc.271.16.9291 CrossRefGoogle Scholar
  38. Umezawa H, Maeda K, Takeuchi T, Okami Y (1966) New antibiotics, bleomycin A and B. J Antibiot 19(5):200–209.  https://doi.org/10.7164/antibiotics.19.200
  39. Vries SJD, Bonvin AMJJ (2011) CPORT: A consensus interface predictor and its performance in prediction-driven docking with HADDOCK. PLoS One 6(3):e17695.  https://doi.org/10.1371/journal.pone.0017695 CrossRefGoogle Scholar
  40. Wang Y, Kendall J, Cavet JS, Giedroc DP (2010) Elucidation of the functional metal binding profile of a CdII/PbII sensor CmtRSc from Streptomyces coelicolor. Biochemistry 49(31):6617–6626.  https://doi.org/10.1021/bi100490u CrossRefGoogle Scholar
  41. Zolotarev A, Unnikrishnan M, Shmukler B, Clark J, Vandorpe D, Grigorieff N, Rubin E, Alper S (2008) Increased sulfate uptake by E. coli overexpressing the SLC26-related SulP protein Rv1739c from Mycobacterium tuberculosis. Comp Biochem Physiol A Mol Integr Physiol 149(3):255–266.  https://doi.org/10.1016/j.cbpa.2007.12.005 CrossRefGoogle Scholar

Copyright information

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

Authors and Affiliations

  • Hong Chen
    • 1
    • 2
  • Junhua Wang
    • 1
  • Jiaqi Cui
    • 1
    • 2
  • Cheng Wang
    • 3
  • Shaoxiong Liang
    • 4
  • Huanhuan Liu
    • 5
  • Jianping Wen
    • 1
    • 2
    Email author
  1. 1.Key Laboratory of Systems Bioengineering (Ministry of Education)Tianjin UniversityTianjinPR China
  2. 2.SynBio Research Platform, Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), School of Chemical Engineering and TechnologyTianjin UniversityTianjinPR China
  3. 3.Department of Forestry Engineering, College of ForestryNorthwest A&F UniversityYanglingPR China
  4. 4.College of Chemical EngineeringHuaqiao UniversityXiamenPR China
  5. 5.Key Laboratory of Food Nutrition and Safety, Ministry of Education, School of Food Engineering and BiotechnologyTianjin University of Science and TechnologyTianjinChina

Personalised recommendations