Applied Microbiology and Biotechnology

, Volume 101, Issue 8, pp 3273–3282 | Cite as

LetR is a TetR family transcription factor from Lysobacter controlling antifungal antibiotic biosynthesis

  • Ping Wang
  • Hongfu Chen
  • Guoliang QianEmail author
  • Fengquan LiuEmail author
Applied genetics and molecular biotechnology


Heat-stable antifungal factor (HSAF) is a newly identified and broad-spectrum antifungal antibiotic from Lysobacter enzymogenes, a ubiquitous environmental proteobacterium. Yet, the regulatory mechanism for HSAF biosynthesis in L. enzymogenes remains poorly understood. Here, we report the identification of a TetR-family protein Le1552 (LetR) from L. enzymogenes strain OH11 that is involved in transcriptional repression of HSAF production. Bacterial one-hybrid and gel mobility shift assays show that LetR directly binds to PHSAF (the promoter region of the HSAF biosynthesis operon). A DNA truncation assay further reveals a core region in PHSAF that is responsible for LetR binding. In-frame deletion of letR in wild-type OH11 is found to significantly increase HSAF levels and key biosynthetic gene transcription, while overexpression of letR in the wild-type background remarkably reduces HSAF levels as well as related gene expression instead. Together, we have identified not only a new regulator for the HSAF biosynthesis but also constructed a higher HSAF-producing deletion strain (ΔletR) of L. enzymogenes, which shall be of great value in promoting HSAF production for pharmaceutical and biological control purposes.


Lysobacter HSAF TetR Regulation Antibiotics 



We thank Prof. Liangcheng Du (University of Nebraska-Lincoln) for the suggestions on manuscript organization. We also thank Prof. Zheng-Guo He from Huazhong Agricultural University (China) for kindly providing the bacterial one-hybrid system. Author contributions: G.Q. and F.L. conceived the project and designed experiments. P.W and H.C carried out experiments. P.W., H. C., G.Q., and F.L. analyzed data. P.W and G.Q. wrote the manuscript draft. F.L. revised the manuscript.

Compliance with ethical standards


This study was supported by National Basic Research (973) program of China (2015CB150600 to G.Q.), the Fundamental Research Funds for the Central Universities (Y0201600126 and KYTZ201403 to G.Q.), Special Fund for Agro-Scientific Research in the Public Interest (no. 201303015 to G.Q. and F.L.), National Natural Science Foundation of China (31371981 and 31572046 to G.Q.) and the Jiangsu Provincial Key Technology Support Program (BE2014386 and BE2015354 to F.L.), the Basal Research Funds from JAAS [ZX(15)1006 to F. L.], Jiangsu Agricultural Science and Technology Innovation Funds [CX(16)1049 to F. L.), ‘948’ Project of the Ministry of Agriculture (2014-Z24 to F.L.), and National pear industry technology system (CARS-29-09 to F.L. and G.Q.).

Conflict of interest

The authors declare that they have no competing interests.

Ethical approval

This article does not contain any studies with human participants or animals.

Supplementary material

253_2017_8117_MOESM1_ESM.pdf (276 kb)
ESM 1 (PDF 275 kb)


  1. Christensen P, Cook FD (1978) Lysobacter, a new genus of nonfruiting, gliding bacteria with a high base ratio. Int J Syst Evol Microbiol 28:367–393. doi: 10.1099/00207713-28-3-367 Google Scholar
  2. de Bruijn I, Cheng X, de Jager VE, Expósito RG, Watrous J, Patel N, Postma J, Dorrestein PC, Kobayashi D, Raaijmakers JM (2015) Comparative genomics and metabolic profiling of the genus Lysobacter. BMC Genomics 16:991. doi: 10.1186/s12864-015-2191-z CrossRefPubMedPubMedCentralGoogle Scholar
  3. Ding YJ, Li YY, Li ZY, Zhang JL, Lu CH, Wang HX, Shen YM, Du LC (2016a) Alteramide B is a microtubule antagonist of inhibiting Candida albicans. Biochim Biophys Acta 1860(10):2097–2106. doi: 10.1016/j.bbagen.2016.06.025 CrossRefPubMedGoogle Scholar
  4. Ding YJ, Li ZY, Li YY, Lu CH, Wang HX, Shen YM, Du LC (2016b) HSAF-induced antifungal effects in Candida albicans through ROS-mediated apoptosis. RSC Adv 6(37):30895–30904. doi: 10.1039/C5RA26092B CrossRefPubMedPubMedCentralGoogle Scholar
  5. Ferluga S, Venturi V (2009) OryR is a LuxR-family protein involved in interkingdom signaling between pathogenic Xanthomonas oryzae pv. oryzae and rice. J Bacteriol 191(3):890–897. doi: 10.1128/JB.01507-08 CrossRefPubMedGoogle Scholar
  6. Guo MM, Feng H, Zhang J, Wang WQ, Wang Y, Li YQ, Gao CH, Chen HC, Feng Y, Hen ZG (2009) Dissecting transcription regulatory pathways through a new bacterial one-hybrid reporter system. Genome Res 19(7):1301–1308. doi: 10.1101/gr.086595.108 CrossRefPubMedPubMedCentralGoogle Scholar
  7. Hayward AC, Fegan N, Fegan M, Stirling GR (2010) Stenotrophomonas and Lysobacter: ubiquitous plant-associated gamma-Proteobacteria of developing significance in applied microbiology. J Appl Microbiol 108(3):756–770. doi: 10.1111/j.1365-2672.2009.04471.x CrossRefPubMedGoogle Scholar
  8. Hoang TT, Karkhoff-Schweizer RR, Kutchma AJ, Schweizer HP (1998) A broad-host-range Flp-FRT recombination system for site-specific excision of chromosomally-located DNA sequences: application for isolation of unmarked Pseudomonas aeruginosa mutants. Gene 212(1):77–86. doi: 10.1016/S0378-1119(98)00130-9 CrossRefPubMedGoogle Scholar
  9. Kobayashi DY, Reedy RM, Palumbo JD, Zhou JM, Yuen GY (2005) A clp gene homologue belonging to the Crp gene family globally regulates lytic enzyme production, antimicrobial activity, and biological control activity expressed by Lysobacter enzymogenes strain C3. Appl Environ Microbiol 71(1):261–269. doi: 10.1128/AEM.71.1.261-269.2005 CrossRefPubMedPubMedCentralGoogle Scholar
  10. 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. doi: 10.1016/0378-1119(95)00584-1 CrossRefPubMedGoogle Scholar
  11. Li S, Du L, Yuen G, Harris SD (2006) Distinct ceramide synthases regulate polarized growth in the filamentous fungus Aspergillus nidulans. Mol Biol Cell 17(3):1218–1227. doi: 10.1091/mbc.E05-06-0533 CrossRefPubMedPubMedCentralGoogle Scholar
  12. Li S, Jochum CC, Yu F, Zaleta-Rivera K, Du L, Harris SD, Yuen GY (2008) An antibiotic complex from Lysobacter enzymogenes strain C3: antimicrobial activity and role in plant disease control. Phytopathology 98(6):695–701. doi: 10.1094/PHYTO-98-6-0695 CrossRefPubMedGoogle Scholar
  13. Li Y, Chen H, Ding Y, Xie Y, Wang H, Cerny RL, Shen YM, Du LC (2014) Iterative assembly of two separate polyketide chains by the same single-module bacterial polyketide synthase in the biosynthesis of HSAF. Angew Chem Int Ed Engl 53(29):7524–7530. doi: 10.1002/anie.201403500 CrossRefPubMedPubMedCentralGoogle Scholar
  14. Lou L, Qian G, Xie Y, Hang J, Chen H, Zaleta-Rivera K, Li YY, Shen YM, Dussault PH, Liu FQ, Du LC (2011) Biosynthesis of HSAF, a tetramic acid-containing macrolactam from Lysobacter enzymogenes. J Am Chem Soc 133(4):643–645. doi: 10.1021/ja105732c CrossRefPubMedGoogle Scholar
  15. Qian GL, Hu BS, Jiang YH, Liu FQ (2009) Identification and characterization of Lysobacter enzymogenes as a biological control agent against some fungal pathogens. Agr Sci China 8(1):68–75CrossRefGoogle Scholar
  16. Qian GL, Wang YS, Qian DY, Fan JQ, Hu BS, Liu FQ (2012) Selection of available suicide vectors for gene mutagenesis using chiA (a chitinase encoding gene) as a new reporter and primary functional analysis of chiA in Lysobacter enzymogenes strain OH11. World J Microbiol Biotechnol 28(2):549–557. doi: 10.1007/s11274-011-0846-8 CrossRefPubMedGoogle Scholar
  17. Qian GL, Wang YL, Liu YR, Xu FF, He YW, Du LC, Venturi V, Fan JQ, Hu BS, Liu FQ (2013) Lysobacter enzymogenes uses two distinct cell-cell signaling systems for differential regulation of secondary-metabolite biosynthesis and colony morphology. Appl Environ Microbiol 79(21):6604–6616. doi: 10.1128/AEM.01841-13 CrossRefPubMedPubMedCentralGoogle Scholar
  18. Qian GL, Xu FF, Venturi V, Du LC, Liu FQ (2014) Roles of a solo LuxR in the biological control agent Lysobacter enzymogenes strain OH11. Phytopathology 104(3):224–231. doi: 10.1094/PHYTO-07-13-0188-R CrossRefPubMedPubMedCentralGoogle Scholar
  19. Ramos JL, Martinez-Bueno M, Molina-Henares A, Teran W, Watanabe K, Zhang XD, Gallegos MT, Brennan R, Tobes R (2005) The TetR family of transcriptional repressors. Microbiol Mol Biol Rev 69(2):326–356. doi: 10.1128/MMBR.69.2.326-356.2005 CrossRefPubMedPubMedCentralGoogle Scholar
  20. Sakamoto K, Agari Y, Kuramitsu S, Shinkai A (2011) Phenylacetyl coenzyme A is an effector molecule of the TetR family transcriptional repressor PaaR from Thermus thermophiles HB8. J Bacteriol 193(17):4388–4395. doi: 10.1128/JB.05203-11 CrossRefPubMedPubMedCentralGoogle Scholar
  21. Stratigopoulos G, Cundliffe E (2002) Expression analysis of the tylosin-biosynthetic gene cluster: pivotal regulatory role of the tylQ gene product. Chem Biol 9(1): 71–78. doi:  org/10.1016/S1074-5521(01)00095-3
  22. Typas A, Becker G, Hengge R (2007) The molecular basis of selective promoter activation by the σS subunite of RNA polymerase. Mol Microbiol 63(5):1296–1306. doi: 10.1111/j.1365-2958.2007.05601.x CrossRefPubMedGoogle Scholar
  23. Walsh C (2003) Antibiotics: actions, origins, resistance. ASM Press, Washington DCCrossRefGoogle Scholar
  24. Wang YS, Zhao YX, Zhang J, Zhao YY, Shen Y, Su ZH, Xu GG, Du LC, Huffman JM, Venturi V, Qian GL, Liu FQ (2014) Transcriptomic analysis reveals new regulatory roles of Clp signaling in secondary metabolite biosynthesis and surface motility in Lysobacter enzymogenes OH11. Appl Microbiol Biotechnol 98(21):9009–9020. doi: 10.1007/s00253-014-6072-1 CrossRefPubMedPubMedCentralGoogle Scholar
  25. Xie YX, Wright S, Shen YM, Du LC (2012) Bioactive natural products from Lysobacter. Nat Prod Rep 29(11):1277–1287. doi: 10.1039/c2np20064c CrossRefPubMedPubMedCentralGoogle Scholar
  26. Xu LX, Wu P, Wright SJ, Du LC, Wei XY (2015) Bioactive polycyclic tetramate macrolactams from Lysobacter enzymogenes and their absolute configurations by theoretical ECD calculations. J Nat Prod 78(8):1841–1847. doi: 10.1021/acs.jnatprod.5b00099 CrossRefPubMedGoogle Scholar
  27. Xu HY, Chen HF, Shen Y, Du LC, Chou SH, Liu HX, Qian GL, Liu FQ (2016) Direct regulation of extracellular chitinase production by the transcription factor LeClp in Lysobacter enzymogenes OH11. Phytopathology 106(9):971–977. doi: 10.1094/PHYTO-01-16-0001-R CrossRefPubMedGoogle Scholar
  28. Yu FG, Zaleta-Rivera K, Zhu XC, Huffman J, Millet JC, Harris SD, Yuen G, Li XC, Du LC (2007) Structure and biosynthesis of heat-stable antifungal factor (HSAF), a broad-spectrum antimycotic with a novel mode of action. Antimicrob Agents Ch 51(1):64–72. doi: 10.1128/AAC.00931-06 CrossRefGoogle Scholar
  29. Zhang Z, Yuen GY (1999) Biological control of Bipolaris sorokiniana on tall fescue by Stenotrophomonas maltophilia strain C3. Phytopathology 89(9):817–822. doi: 10.1094/PHYTO.1999.89.9.817 CrossRefPubMedGoogle Scholar
  30. Zhang W, Li YY, Qian GL, Wang Y, Chen HT, Li YZ, Liu FQ, Shen YM, Du LC (2011) Identification and characterization of the anti-methicillin-resistant Staphylococcus aureus WAP-8294A2 biosynthetic gene cluster from Lysobacter enzymogenes OH11. Antimicrob Agents Ch 55(12):5581–5589. doi: 10.1128/AAC.05370-11 CrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2017

Authors and Affiliations

  1. 1.College of Plant ProtectionNanjing Agricultural UniversityJiangsu ProvincePeople’s Republic of China
  2. 2.Institute of Plant ProtectionJiangsu Academy of Agricultural SciencesNanjingPeople’s Republic of China

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