Spermidine plays a significant role in stabilizing a master transcription factor Clp to promote antifungal activity in Lysobacter enzymogenes

  • Yun Zhao
  • Tingting Zhang
  • Yang Ning
  • Danyu Shen
  • Nianda Yang
  • Yingying Li
  • Shan-Ho Chou
  • Liang Yang
  • Guoliang QianEmail author
Applied genetics and molecular biotechnology


Spermidine is a common polyamine compound produced in bacteria, but its roles remain poorly understood. The bacterial SpeD encodes an S-adenosylmethionine decarboxylase that participates in spermidine synthesis. Lysobacter enzymogenes is an efficient environmental predator of crop fungal pathogens by secreting an antifungal antibiotic HSAF (heat-stable antifungal factor), while Clp is a master transcription factor essential for the antifungal activity of L. enzymogenes. In this work, we observed that speD was a close genomic neighbor of the clp gene. This genomic arrangement also seems to occur in many other bacteria, but the underlying reason remains unclear. By using L. enzymogenes OH11 as a working model, we showed that SpeD was involved in spermidine production that was essential for the L. enzymogenes antifungal activity. Spermidine altered the bacterial growth capability and HSAF production, both of which critically contributed to the L. enzymogenes antifungal activity. We further found that spermidine in L. enzymogenes was able to play a crucial, yet indirect role in maintaining the Clp level in vivo, at least partially accounting for its role in the antifungal activity. Thus, our findings suggested that spermidine probably plays an uncharacterized role in maintaining the levels of the master transcription regulator Clp to optimize its role in antifungal activity in an agriculturally beneficial bacterium.


Lysobacter Polyamine Spermidine Antifungal activity Clp 



We thank Prof. Wei Qian from the Chinese Academy of Science for providing facilities in MST assay and the anti-Clp antibody.

Author contribution

S.C., L.Y., and G.Q. conceived the project and designed experiments. Y.Z., K.X., T.Z., D.S., S.H., Y.H., and L.Y. carried out experiments. Z.Y., S.D., L.Y., S.C, and G.Q. analyzed data. Z.Y., S.C., L.Y., and G.Q. wrote and revised the manuscript draft.

Funding information

This study was supported by the National Key Research and Development Program (2017YFD0201100 to G. Qian), the Fundamental Research Funds for the Central Universities (KYT201805, Y0201600126, and KYTZ201403 to GQ), the Jiangsu Agricultural Science and Technology Innovation Fund (CX(18)1003), Natural Science Fund of Jiangsu Province (BK20181325), the Innovation Team Program for Jiangsu Universities (2017), the National Ministry of Science and Technology of Taiwan (105-2113-M-005-013-MY2 to S. Chou), and the AcRF Tier 2 (MOE2016-T2-1-010 to L. Yang) from the Ministry of Education, Singapore. The funders have no role in study design.

Compliance with ethical standards

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_2018_9596_MOESM1_ESM.pdf (427 kb)
ESM 1 (PDF 426 kb)
253_2018_9596_MOESM2_ESM.xlsx (148 kb)
ESM 2 (XLSX 148 kb)


  1. Anders S, Huber W (2010) Differential expression analysis for sequence count data. Genome Biol 11:R106. CrossRefGoogle Scholar
  2. Chen Y, Xia J, Su Z, Xu G, Gomelsky M, Qian G, Liu F (2017) The regulator of type IV pili synthesis, PilR, from Lysobacter controls antifungal antibiotic production via a c-di-GMP pathway. Appl Environ Microbiol 83:e03397–e03316. Google Scholar
  3. 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 396:646–662. CrossRefGoogle Scholar
  4. 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. Google Scholar
  5. Di Martino ML, Campilongo R, Casalino M, Micheli G, Colonna B, Prosseda G (2013) Polyamines: emerging players in bacteria-host interactions. Int J Med Microbiol 303:484–491. CrossRefGoogle Scholar
  6. Gentleman RC, Carey VJ, Bates DM, Bolstad B, Dettling M, Dudoit S, Ellis B, Gautier L, Ge Y, Gentry J, Hornik K, Hothorn T, Huber W, Iacus S, Irizarry R, Leisch F, Li C, Maechler M, Rossini AJ, Sawitzki G, Smith C, Smyth G, Tierney L, Yang JY, Zhang J (2004) Bioconductor: open software development for computational biology and bioinformatics. Genome Biol 5:R80. CrossRefGoogle Scholar
  7. Gevrekci AÖ (2017) The roles of polyamines in microorganisms. World J Microbiol Biotechnol 33:204. CrossRefGoogle Scholar
  8. Goytia M, Dhulipala VL, Shafer WM (2013) Spermidine impairs biofilm formation by Neisseria gonorrhoeae. FEMS Microbiol Lett 343:64–69. CrossRefGoogle Scholar
  9. Hamana K, Matsuzaki S (1992) Polyamines as a chemotaxonomic marker in bacterial systematics. Crit Rev Microbiol 18:261–283. CrossRefGoogle Scholar
  10. 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 212:77–86. CrossRefGoogle Scholar
  11. Hobley L, Li B, Wood JL, Kim SH, Naidoo J, Ferreira AS, Khomutov M, Khomutov A, Stanley-Wall NR, Michael AJ (2017) Spermidine promotes Bacillus subtilis biofilm formation by activating expression of the matrix regulator slrR. J Biol Chem 292:12041–12053. CrossRefGoogle Scholar
  12. Igarashi K, Kashiwagi K (2006) Polyamine modulon in Escherichia coli: genes involved in the stimulation of cell growth by polyamines. J Biochem 139:11–16. CrossRefGoogle Scholar
  13. Kara DA, Borzova VA, Markossian KA, Kleymenov SY, Kurganov BI (2017) A change in the pathway of dithiothreitol-induced aggregation of bovine serum albumin in the presence of polyamines and arginine. Int J Biol Macromol 104:889–899. CrossRefGoogle Scholar
  14. 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 71:261–269. CrossRefGoogle Scholar
  15. 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 166:175–176. CrossRefGoogle Scholar
  16. Li S, Du L, Yuen G, Harris SD (2006) Distinct ceramide synthases regulate polarized growth in the filamentous fungus Aspergillus nidulans. Mol Biol Cell 17:1218–1227. CrossRefGoogle Scholar
  17. 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 98:695–701. CrossRefGoogle Scholar
  18. 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 133:643–645. CrossRefGoogle Scholar
  19. McGinnis MW, Parker ZM, Walter NE, Rutkovsky AC, Cartaya-Marin C, Karatan E (2009) Spermidine regulates Vibrio cholera biofilm formation via transport and signaling pathways. FEMS Microbiol Lett 299:166–174. CrossRefGoogle Scholar
  20. Pegg AE, Shantz LM (1998) Assay of mammalian S-adenosylmethionine decarboxylase activity. Mol Biol 79:45–49Google Scholar
  21. 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
  22. 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. CrossRefGoogle Scholar
  23. 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. CrossRefGoogle Scholar
  24. 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. CrossRefGoogle Scholar
  25. Sobe RC, Bond WG, Wotanis CK, Zayner JP, Burriss MA, Fernandez N, Bruger EL, Wasters CM, Neufeld HS (2017) Karatan E (2017) Spermine inhibits Vibrio cholerae biofilm formation through the NspS-MbaA polyamine signaling system. J Biol Chem 292:17025–17036. CrossRefGoogle Scholar
  26. Su Z, Chen H, Wang P, Tombosa S, Du L, Han Y, Shen Y, Qian G, Liu F (2017) 4-Hydroxybenzoic acid is a diffusible factor that connects metabolic shikimate pathway to the biosynthesis of a unique antifungal metabolite in Lysobacter enzymogenes. Mol Microbiol 104:163–178. CrossRefGoogle Scholar
  27. Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S (2011) MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol 28:2731–2739. CrossRefGoogle Scholar
  28. Vega AD, Delcour AH (1996) Polyamines decrease Escherichia coli outer membrane permeability. J Bacteriol 178:3715–3721Google Scholar
  29. Wang P, Chen H, Qian G, Liu F (2017) LetR is a TetR family transcription factor from Lysobacter controlling antifungal antibiotic biosynthesis. Appl Microbiol Biotechnol 101:3273–3282. CrossRefGoogle Scholar
  30. Wang Y, Kim SH, Natarajan R, Heindl JE, Bruger EL, Waters CM, Michael AJ, Fuqua C (2016) Spermidine inversely influences surface interactions and planktonic growth in Agrobacterium tumefaciens. J Bacteriol 198:2682–2691. CrossRefGoogle Scholar
  31. Wang YS, Zhao YX, Zhang J, Zhao YY, Shen Y, Su ZH, Xu GG, Du LC, Huffman JM, Venturi V, Qian GG, 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 98:9009–9020. CrossRefGoogle Scholar
  32. Wu D, Lim SC, Dong Y, Wu J, Tao F, Zhou L, Zhang LH, Song H (2012) Structural basis of substrate binding specificity revealed by the crystal structures of polyamine receptors SpuD and SpuE from Pseudomonas aeruginosa. J Miol Biol 416:697–712.
  33. Xie YX, Wright S, Shen YM, Du LC (2012) Bioactive natural products from Lysobacter. Nat Prod Rep 29:1277–1287. CrossRefGoogle Scholar
  34. Xu G, Han S, Huo C, Chin KH, Chou SH, Gomelsky M, Qian G, Liu F (2018) Signaling specificity in the c-di-GMP-dependent network regulating antibiotic synthesis in Lysobacter. Nucleic Acids Res 46:9276–9288. CrossRefGoogle Scholar
  35. Xu H, Chen H, Shen Y, Du L, Chou SH, Liu H, Qian G, Liu F (2016) Direct regulation of extracellular chitinase production by the transcription factor LeClp in Lysobacter enzymogenes OH11. Phytopathology 106:971–977. CrossRefGoogle Scholar
  36. Yin ZY, Liu HQ, Li ZP, Ke XW, Dou DL, Gao XN, Song N, Dai QQ, Wu YX, Kang ZS, Huang LL (2015) Genome sequence of Valsa canker pathogens uncovers a potential adaptation of colonization of woody bark. New Phytol 208:1202–1216. CrossRefGoogle Scholar
  37. 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 51:64–72. CrossRefGoogle Scholar

Copyright information

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

Authors and Affiliations

  1. 1.College of Plant Protection (Key Laboratory of Integrated Management of Crop Diseases and Pests)Nanjing Agricultural UniversityNanjing CityPeople’s Republic of China
  2. 2.Singapore Centre for Environmental Life Sciences EngineeringNanyang Technological UniversitySingaporeSingapore
  3. 3.Institute of Biochemistry, and NCHU Agricultural Biotechnology CenterNational Chung Hsing UniversityTaichungTaiwan, Republic of China

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