Applied Microbiology and Biotechnology

, Volume 99, Issue 2, pp 801–811 | Cite as

Identification of a small molecule signaling factor that regulates the biosynthesis of the antifungal polycyclic tetramate macrolactam HSAF in Lysobacter enzymogenes

  • Yong Han
  • Yan Wang
  • Simon Tombosa
  • Stephen Wright
  • Justin Huffman
  • Gary Yuen
  • Guoliang Qian
  • Fengquan Liu
  • Yuemao Shen
  • Liangcheng Du
Applied genetics and molecular biotechnology


Lysobacter species are emerging as new sources of antibiotics. The regulation of these antibiotics is not well understood. Here, we identified a small molecule metabolite (LeDSF3) that regulates the biosynthesis of the antifungal antibiotic heat-stable antifungal factor (HSAF), a polycyclic tetramate macrolactam with a structure and mode of action distinct from the existing antifungal drugs. LeDSF3 was isolated from the culture broth of Lysobacter enzymogenes, and its chemical structure was established by NMR and MS. The purified compound induced green fluorescence in a reporter strain of Xanthomonas campestris, which contained a gfp gene under the control of a diffusible signaling factor (DSF)-inducible promoter. Exogenous addition of LeDSF3 in L. enzymogenes cultures significantly increased the HSAF yield, the transcription of HSAF biosynthetic genes, and the antifungal activity of the organism. The LeDSF3-regulated HSAF production is dependent on the two-component regulatory system RpfC/RpfG. Moreover, LeDSF3 upregulated the expression of the global regulator cAMP receptor-like protein (Clp). The disruption of clp led to no HSAF production. Together, the results show that LeDSF3 is a fatty acid-derived, diffusible signaling factor positively regulating HSAF biosynthesis and that the signaling is mediated by the RfpC/RpfG-Clp pathway. These findings may facilitate the antibiotic production through applied genetics and molecular biotechnology in Lysobacter, a group of ubiquitous yet underexplored microorganisms.


Diffusible signaling factor Natural product biosynthesis Regulation HSAF Lysobacter enzymogenes 



This work was supported in part by the 973 Project (2013CB734002, 2015CB150602), the NIH (R01AI097260), and Program for Changjiang Scholars and Innovative Research Team in University (IRT13028). We thank Prof. Lindow for the generous gift of DSF reporter strain X. campestris 8523/pKLN55. Ron Cerny, Martha Morton, Kurt Wulser, and Javier Seravalli are thanked for technical assistance.

Supplementary material

253_2014_6120_MOESM1_ESM.pdf (424 kb)
ESM 1 (PDF 423 kb)


  1. Almeida RP, Killiny N, Newman KL, Chatterjee S, Ionescu M, Lindow SE (2012) Contribution of rpfB to cell-to-cell signal synthesis, virulence, and vector transmission of Xylella fastidiosa. Mol Plant Microbe Interact 25:453–462CrossRefPubMedGoogle Scholar
  2. Amari DT, Marques CN, Davies DG (2013) The putative enoyl-coenzyme A hydratase DspI is required for production of the Pseudomonas aeruginosa biofilm dispersion autoinducer cis-2-decenoic acid. J Bacteriol 195:4600–4610CrossRefPubMedPubMedCentralGoogle Scholar
  3. Barber CE, Tang JL, Feng JX, Pan MQ, Wilson TJ, Slater H, Dow JM, Williams P, Daniels MJ (1997) A novel regulatory system required for pathogenicity of Xanthomonas campestris is mediated by a small diffusible signal molecule. Mol Microbiol 24:555–566CrossRefPubMedGoogle Scholar
  4. Bi H, Christensen QH, Feng Y, Wang H, Cronan JE (2012) The Burkholderia cenocepacia BDSF quorum sensing fatty acid is synthesized by a bifunctional crotonase homologue having both dehydratase and thioesterase activities. Mol Microbiol 83:840–855CrossRefPubMedPubMedCentralGoogle Scholar
  5. Boon C, Deng Y, Wang LH, He Y, Xu JL, Fan Y, Pan SQ, Zhang LH (2008) A novel DSF-like signal from Burkholderia cenocepacia interferes with Candida albicans morphological transition. ISME J 2:27–36CrossRefPubMedGoogle Scholar
  6. Case RJ, Labbate M, Kjelleberg S (2008) AHL-driven quorum-sensing circuits: their frequency and function among the Proteobacteria. ISME J 2:345–349CrossRefPubMedGoogle Scholar
  7. Chatterjee S, Wistrom C, Lindow SE (2008) A cell-cell signaling sensor is required for virulence and insect transmission of Xylella fastidiosa. Proc Natl Acad Sci U S A 105:2670–2675CrossRefPubMedPubMedCentralGoogle Scholar
  8. Cheng Z, He YW, Lim SC, Qamra R, Walsh MA, Zhang LH, Song H (2010) Structural basis of the sensor-synthase interaction in autoinduction of the quorum sensing signal DSF biosynthesis. Structure 18:1199–1209CrossRefPubMedGoogle Scholar
  9. Chin KH, Lee YC, Tu ZL, Chen CH, Tseng YH, Yang JM, Ryan RP, McCarthy Y, Dow JM, Wang AH, Chou SH (2010) The cAMP receptor-like protein CLP is a novel c-di-GMP receptor linking cell-cell signaling to virulence gene expression in Xanthomonas campestris. J Mol Biol 396:646–662CrossRefPubMedGoogle Scholar
  10. Christensen P, Cook FD (1978) Lysobacter, a new genus of non-fruiting, gliding bacteria with a high base ratio. Int J Syst Bacteriol 28:367–393CrossRefGoogle Scholar
  11. Comella N, Grossman AD (2005) Conservation of genes and processes controlled by the quorum response in bacteria: characterization of genes controlled by the quorum-sensing transcription factor ComA in Bacillus subtilis. Mol Microbiol 57:1159–1174CrossRefPubMedGoogle Scholar
  12. Corre C, Song L, O’Rourke S, Chater KF, Challis GL (2008) 2-Alkyl-4-hydroxymethylfuran-3-carboxylic acids, antibiotic production inducers discovered by Streptomyces coelicolor genome mining. Proc Natl Acad Sci U S A 105:17510–17515CrossRefPubMedPubMedCentralGoogle Scholar
  13. Deng Y, Boon C, Eberl L, Zhang LH (2009) Differential modulation of Burkholderia cenocepacia virulence and energy metabolism by the quorum-sensing signal BDSF and its synthase. J Bacteriol 191:7270–7278CrossRefPubMedPubMedCentralGoogle Scholar
  14. Deng Y, Wu J, Eberl L, Zhang LH (2010) Structural and functional characterization of diffusible signal factor family quorum-sensing signals produced by members of the Burkholderia cepacia complex. Appl Environ Microbiol 76:4675–4683CrossRefPubMedPubMedCentralGoogle Scholar
  15. Deng Y, Wu J, Tao F, Zhang LH (2011) Listening to a new language: DSF-based quorum sensing in Gram-negative bacteria. Chem Rev 111:160–173CrossRefPubMedGoogle Scholar
  16. Dusane DH, Matkar P, Venugopalan VP, Kumar AR, Zinjarde SS (2011) Cross-species induction of antimicrobial compounds, biosurfactants and quorum-sensing inhibitors in tropical marine epibiotic bacteria by pathogens and biofouling microorganisms. Curr Microbiol 62:974–980CrossRefPubMedGoogle Scholar
  17. Fuqua C, Greenberg EP (2002) Listening in on bacteria: acyl-homoserine lactone signaling. Nat Rev Mol Cell Biol 3:685–695CrossRefPubMedGoogle Scholar
  18. Galloway WR, Hodgkinson JT, Bowden SD, Welch M, Spring DR (2011) Quorum sensing in Gram-negative bacteria: small-molecule modulation of AHL and AI-2 quorum sensing pathways. Chem Rev 111:28–67CrossRefPubMedGoogle Scholar
  19. He YW, Wang C, Zhou L, Song H, Dow JM, Zhang LH (2006) Dual signaling functions of the hybrid sensor kinase RpfC of Xanthomonas campestris involve either phosphorelay or receiver domain-protein interaction. J Biol Chem 281:33414–33421CrossRefPubMedGoogle Scholar
  20. He YW, Wu J, Cha JS, Zhang LH (2010) Rice bacterial blight pathogen Xanthomonas oryzae pv. oryzae produces multiple DSF-family signals in regulation of virulence factor production. BMC Microbiol 10:187CrossRefPubMedPubMedCentralGoogle Scholar
  21. He YW, Wu J, Zhou L, Yang F, He YQ, Jiang BL, Bai L, Xu Y, Deng Z, Tang JL, Zhang LH (2011) Xanthomonas campestris diffusible factor is 3-hydroxybenzoic acid and is associated with xanthomonadin biosynthesis, cell viability, antioxidant activity, and systemic invasion. Mol Plant Microbe Interact 24:948–957CrossRefPubMedGoogle Scholar
  22. Horinouchi S, Beppu T (1994) A-factor as a microbial hormone that controls cellular differentiation and secondary metabolism in Streptomyces griseus. Mol Microbiol 12:859–864CrossRefPubMedGoogle Scholar
  23. Hou J, Robbel L, Marahiel MA (2011) Identification and characterization of the lysobactin biosynthetic gene cluster reveals mechanistic insights into an unusual termination module architecture. Chem Biol 18:655–664CrossRefPubMedGoogle Scholar
  24. Huang TP, Wong ACL (2007) Extracellular fatty acids facilitate flagella-independent translocation by Stenotrophomonas maltophilia. Res Microbiol 158:702–711CrossRefPubMedGoogle Scholar
  25. Khoklov AS, Tovarova II, Borisova LN, Pliner SA, Schevchenko LA, Kornitskaya EY, Ivkina NS, Rapoport IA (1967) A-factor responsible for the biosynthesis of streptomycin by a mutant strain of Actinomyces streptomycini. Dokl Akad Nauk SSSR 177:232–235Google Scholar
  26. 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 71:261–269CrossRefPubMedPubMedCentralGoogle Scholar
  27. Li S, Du L, Yuen G, Harris SD (2006) Distinct ceramide synthases regulate polarized growth in the filamentous fungus Aspergillus nidulans. Mol Biol Cell 17:1218–1227CrossRefPubMedPubMedCentralGoogle Scholar
  28. Li Y, Huffman J, Li Y, Du L, Shen Y (2012) 3-Hydroxylation of the polycyclic tetramate macrolactam in the biosynthesis of antifungal HSAF from Lysobacter enzymogenes C3. Med Chem Comm 9:982–986CrossRefGoogle Scholar
  29. Li Y, Chen H, Ding Y, Xie Y, Wang H, Cerny RL, Shen Y, Du L (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 53:7524–7530CrossRefGoogle Scholar
  30. Liu G, Chater KF, Chandra G, Niu G, Tan H (2013) Molecular regulation of antibiotic biosynthesis in Streptomyces. Microbiol Mol Biol Rev 77:112–143CrossRefPubMedPubMedCentralGoogle Scholar
  31. Lou L, Qian G, Xie Y, Hang J, Chen H, Zaleta-Rivera K, Li Y, Shen Y, Dussault PH, Liu F, Du L (2011) Biosynthesis of HSAF, a tetramic acid-containing macrolactam from Lysobacter enzymogenes. J Am Chem Soc 133:643–645CrossRefPubMedGoogle Scholar
  32. Lou L, Chen H, Cerny RL, Li Y, Shen Y, Du L (2012) Unusual activities of the thioesterase domain for the biosynthesis of the polycyclic tetramate macrolactam HSAF in Lysobacter enzymogenes C3. Biochem 51:4–6CrossRefGoogle Scholar
  33. Miller MB, Bassler BL (2001) Quorum sensing in bacteria. Annu Rev Microbiol 55:165–199CrossRefPubMedGoogle Scholar
  34. Newman KL, Almeida RP, Purcell AH, Lindow SE (2004) Cell-cell signaling controls Xylella fastidiosa interactions with both insects and plants. Proc Natl Acad Sci U S A 101:1737–1742CrossRefPubMedPubMedCentralGoogle Scholar
  35. Qian G, Wang Y, Liu Y, Xu F, He YW, Du L, Venturi V, Fan J, Hu B, Liu F (2013a) Lysobacter enzymogenes uses two distinct cell-cell signaling systems for differential regulation of secondary-metabolite biosynthesis and colony morphology. Appl Environ Microbiol 79:6604–6616CrossRefPubMedPubMedCentralGoogle Scholar
  36. Qian G, Zhou Y, Zhao Y, Song Z, Wang S, Fan J, Hu B, Venturi V, Liu F (2013b) Proteomic analysis reveals novel extracellular virulence-associated proteins and functions regulated by the diffusible signal factor (DSF) in Xanthomonas oryzae pv. oryzicola. J Proteome Res 12:3327–3341CrossRefPubMedGoogle Scholar
  37. Ryan RP, Dow JM (2011) Communication with a growing family: diffusible signal factor (DSF) signaling in bacteria. Trends Microbiol 19:145–152CrossRefPubMedGoogle Scholar
  38. Sarpe VA, Kulkarni SS (2011) Synthesis of maradolipid. J Org Chem 76:6866–6870CrossRefPubMedGoogle Scholar
  39. Schuster M, Sexton DJ, Diggle SP, Greenberg EP (2013) Acyl-homoserine lactone quorum sensing: from evolution to application. Annu Rev Microbiol 67:43–63CrossRefPubMedGoogle Scholar
  40. Simionato AVC, da Silva DS, Lambais MR, Carrilho E (2007) Characterization of a putative Xylella fastidiosa diffusible signal factor by HRGC-EI-MS. J Mass Spectrom 42:490–496CrossRefGoogle Scholar
  41. Slater H, Alvarez-Morales A, Barber CE, Daniels MJ, Dow JM (2000) A two-component system involving an HD-GYP domain protein links cell-cell signalling to pathogenicity gene expression in Xanthomonas campestris. Mol Microbiol 38:986–1003CrossRefPubMedGoogle Scholar
  42. Sullivan RF, Holtman MA, Zylstra GJ, White JF, Kobayashi DY (2003) Taxonomic positioning of two biological control agents for plant diseases as Lysobacter enzymogenes based on phylogenetic analysis of 16S rDNA, fatty acid composition and phenotypic characteristics. J Appl Microbiol 94:1079–1086CrossRefPubMedGoogle Scholar
  43. Wang LH, He Y, Gao Y, Wu JE, Dong YH, He C, Wang SX, Wang LX, Xu JL, Tay L, Fang RX, Zhang LH (2004) A bacterial cell-cell communication signal with cross-kingdom structural analogues. Mol Microbiol 51:903–912CrossRefPubMedGoogle Scholar
  44. Wang Y, Qian G, Li Y, Wright S, Shen Y, Liu F, Du L (2013a) Biosynthetic mechanism for sunscreens of the biocontrol agent Lysobacter enzymogenes. PLoS One 8:e66633CrossRefPubMedPubMedCentralGoogle Scholar
  45. Wang Y, Qian G, Liu F, Li YZ, Shen Y, Du L (2013b) Facile method for site-specific gene integration in Lysobacter enzymogenes for yield improvement of the anti-MRSA antibiotics WAP-8294A and the antifungal antibiotic HSAF. ACS Synth Biol 2:670–678CrossRefPubMedGoogle Scholar
  46. Winzer K, Hardie KR, Williams P (2003) LuxS and autoinducer-2: their contribution to quorum sensing and metabolism in bacteria. Adv Appl Microbiol 53:291–396CrossRefPubMedGoogle Scholar
  47. Xie Y, Wright S, Shen Y, Du L (2012) Bioactive natural products from Lysobacter. Nat Prod Rep 19:1277–1287CrossRefGoogle Scholar
  48. Yu F, Zaleta-Rivera K, Zhu X, Huffman J, Millet JC, Harris SD, Yuen G, Li XC, Du L (2007) Structure and biosynthesis of heat-stable antifungal factor (HSAF), a broad-spectrum antimycotic with a novel mode of action. Antimicrob Agents Chemother 51:64–72CrossRefPubMedGoogle Scholar
  49. Yuen GY, Zhang Z (2001) Control of brown patch using the bacterium Stenotrophomonas maltophilia C3 and culture fluid. Int Turfgrass Soc Res J 9:742–747Google Scholar
  50. Yuen GY, Steadman JR, Lindgren DT, Schaff D, Jochum CC (2001) Bean rust biological control using bacterial agents. Crop Prot 20:395–402CrossRefGoogle Scholar
  51. Zhang Z, Yuen GY (1999) Biological control of Bipolaris sorokiniana on tall fescue by Stenotrophomonas maltophilia strain C3. Phytopath 89:817–822CrossRefGoogle Scholar
  52. Zhang W, Li Y, Qian G, Wang Y, Chen H, Li YZ, Liu F, Shen Y, Du L (2011) Identification and characterization of the anti-methicillin-resistant Staphylococcus aureus WAP-8294A2 biosynthetic gene cluster from Lysobacter enzymogenes OH11. Antimicrob Agents Chemother 55:5581–5589CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2014

Authors and Affiliations

  • Yong Han
    • 1
    • 2
  • Yan Wang
    • 1
    • 5
  • Simon Tombosa
    • 1
  • Stephen Wright
    • 1
  • Justin Huffman
    • 1
  • Gary Yuen
    • 3
  • Guoliang Qian
    • 4
  • Fengquan Liu
    • 4
  • Yuemao Shen
    • 2
  • Liangcheng Du
    • 1
  1. 1.Department of ChemistryUniversity of Nebraska—LincolnLincolnUSA
  2. 2.Key Laboratory of Chemical Biology, School of Pharmaceutical SciencesShandong UniversityJinanChina
  3. 3.Department of Plant PathologyUniversity of Nebraska—LincolnLincolnUSA
  4. 4.College of Plant ProtectionNanjing Agricultural UniversityNanjingChina
  5. 5.College of Marine Life SciencesOcean University of ChinaQingdaoChina

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